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Abstract:

A mutant of a potentially therapeutic anti-CD40 antibody is provided
which mutant has reduced ADCC and CDC activities designed to be optimized
as a pharmaceutical agent. A mutant of an agonistic anti-CD40 antibody,
comprising mutation and/or substitution of at least one amino acid in the
constant region to reduce the ADCC and/or CDC activities therein, and a
mutant of an antagonistic anti-CD40 antibody, comprising at least one
mutation or substitution in the constant region to reduce the ADCC and/or
CDC activities therein, both mutants having at least a hinge region
derived from a human IgG2.

Claims:

1. An isolated monoclonal antibody that binds human CD40 and consists of
two heavy chains, each consisting of an amino acid sequence ranging from
Q at position 27 to K at position 474 of SEQ ID NO: 140, and two light
chains, each consisting of an amino acid sequence raging from A at
position 23 to C at position 235 of SEQ ID No: 142.

2. A pharmaceutical composition that comprises the monoclonal antibody
according to claim 1 as an active ingredient.

3. A method of treating or preventing autoimmune disease that comprises
administering the monoclonal antibody according to claim 1 to a patient.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.
10/584,345, filed Feb. 26, 2007, which is a U.S. National Phase of
International Application PCT/JP2004/019750, filed Dec. 24, 2004, which
was published on Jul. 14, 2005, as WO 2005/063981, which claims the
benefit of JP Application No. 2003-431408, filed Dec. 25, 2003, all of
which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

[0002] The present invention relates to an anti-CD40 antibody which
recognizes CD40 which is a type of cell membrane molecules associated
with immunity. Further, the present invention relates to an antibody with
a mutation in the constant region of the human antibody or with a
subclass having its portion substituted in order to decrease an ADCC
and/or CDC activity, while keeping an agonistic or antagonistic activity.

BACKGROUND ART

1. CD40

[0003] CD40 is an antigen having a molecular weight of 50 kDa which is
present on the surface of cell membrane, and expressed in B cells,
dendritic cells (DCs), some types of cancer cells, and thymic epithelial
cells. CD40 is known to play an important role in proliferation and
differentiation of B cells and DCs. CD40 was identified as an antigen
expressed on the surface of human B cells (E. A. Clark et al., Proc.
Natl. Acad. Sci. USA 83: 4494, 1986; and I. Stamenkovic et al., EMBO J.
8: 1403, 1989) and has been considered as a member of the TNF receptor
family which includes low-affinity NGF receptors, TNF receptors, CD27,
OX40 and CD30. Ligands (CD40Ls) to human and murine CD40s have been
recently cloned and found to be membrane proteins type II and expressed
in activated CD4+T cells. CD40L has been also found to introduce strong
signals for activation into human or murine B cells.

[0004] In dendritic cells, CD40 has been observed to be more highly
expressed than in B cells, and it has become clear that CD40 plays an
important role in dendritic cells. Binding of CD40 to CD40L activates
antigen presenting cells (APCs), that is, expresses costimulator
molecules such as CD80 (B7-1) and CD86 (B7-2) or enhances production of
IL-2 (Caux, C., et al.: Activation of human dendritic cells through CD40
cross-linking. J. Exp. Med., 180: 1263, 1994; and Shu, U., et al.:
Activated T cells induce interleukin-12 production by monocyte via
CD40-CD40 ligand interaction. Eur. J. Immunol., 25: 1125, 1995).
Dendritic cells have a strong antigen-presenting capacity and a strong
capacity to activate helper T (Th) cells. Dendritic cells are also
believed to control differentiation of naive Th cells into Th1 or Th2
cells. When peripheral blood monocytes, which are myeloid dendritic
cells, are cultured in the presence of GM-CSF and IL-4, and matured by
CD40L, the resulting matured dendritic cells (DC1) can produce IL-12 in
vitro, and stimulate and activate allogeneic naive Th cells to induce
IFNγ-producing T cells (i.e., to promote their differentiation into
Th1). This action is inhibited by anti-IL-12 antibody and hence may be
effected via IL-12. On the other hand, when plasmacytoid T cells, which
are present in lymphoid T regions and peripheral blood, are cultured in
the presence of IL-3 and CD40 ligand, the resulting lymphoid dendritic
cells (DC2) are shown to be unable to produce IL-12, and stimulate and
activate allogeneic naive Th cells to induce IL-4-producing T cells,
which indicates promotion of their differentiation into Th2. It is
believed that Th1 cells are involved in activation of cellular immunity,
while Th2 cells are associated with enhancement of humoral immunity as
well as restriction of cellular immunity. When cytotoxic T cells (CTL)
are activated with the help of Th1 cells, they may eliminate pathogens (a
number of types of virus, listeria, tuberculosis bacteria, toxoplasma
protozoa, etc.) growing in the cytoplasm and tumor cells.

[0005] Monoclonal anti-CD40 antibodies, which recognize CD40 expressed on
the membrane surface, have been demonstrated to have different biological
activities to B cells. Monoclonal anti-CD40 antibodies are generally
classified into agonistic or antagonistic antibodies against the
interaction between CD40 and CD40L.

[0007] The anti-CD40 antibody has been demonstrated to mature DC (Z. H.
Zhou et. al., Hybridoma, 18: 471, 1999). Furthermore, the role of CD4 T
cells in priming antigen-specific CD8 T cells was reported to be in
activation of DC via CD40-CD40L signaling, and the anti-CD40 monoclonal
antibody (mAb) has been found to be able to replace CD40 helper T cells
in activation of dendritic cells (DC) (Shoenberger, S. P., et. al.:
T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L
interactions. Nature, 480, 1998). Also, administration of an anti-CD40
antibody in mice has been found to be able to protect the animal body
from CD40-expressing tumor cells as well as CD40-non-expressing tumor
cells (French, R. R., et. al.: CD40 antibody evokes a cytotoxic T-cell
response that eradicates lymphoma and bypasses T-cell help. Nature
Medicine, 5, 1999).

[0008] Agonistic anti-CD40 antibodies are expected to be effective for
treatment of infectious diseases, due to bacteria, virus, etc., cancer
and others, based on their functions described above. Anti-CD40
antibodies with superior agonistic activities are described in WO
02/088186. The representative examples of those agonistic antibodies are
KM341-1-19 and 2105 antibodies. The hybridoma KM341-1-19 producing the
KM341-1-19 antibody and the hybridoma 2105 producing the 2105 antibody
were submitted on 27, Sep. 2001 and 17, Apr. 2002, respectively, for
international deposit under the Budapest Treaty, to International Patent
Organisms Depositary, National Institute of Advanced Industrial Science
and Technology (central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan).
Their accession numbers are FERM BP-7759 (KM341-1-19) and FERM BP-8024
(2105).

3. Antagonistic Antibodies

[0009] Taking it in consideration, on the other hand, that CD40 plays an
important role in immunologic responses, as aforementioned, it is
expected that inhibition of binding of CD40 to its ligands would lead to
development of therapeutic agents for immune suppression in organ
transplantation and autoimmune diseases. Sawada, Hase and others have
reported that the peripheral blood of patients suffering from Crohn's
disease has a higher percentage of monocytes highly expressing CD40.
However, such antibodies have not been well known yet as inhibit binding
of CD40 to its ligands. Those inhibitory antibodies would be useful in
functional analysis of CD40 and treatment of diseases requiring
activation of CD40. Inhibitory antibodies to CD40 ligands are also
suggested to be effective against diseases involving binding of CD40 to
the CD40 ligands. However, CD40L was reported to be expressed in
activated platelets (V. Henn et al., Nature 391: 591, 1998), and if an
anti-CD40L antibody is used as a therapeutic agent, thrombus formation
may occur reportedly (T. Kawai et al., Nat. Med. 6: 114, 2000). From this
point of view, antibodies to CD40 are expected to be safer rather than
anti-CD40L antibodies as therapeutic antibody agent to inhibit binding of
CD40 to its ligands. Anti-CD40 antibodies would be required to inhibit
binding of CD40L to CD40 and still not activate CD40 in themselves.

[0010] Such antagonistic anti-CD40 antibodies may be used for treatment of
autoimmune diseases and suppression of immunologic rejections in
transplantation of organs, bone marrow, etc., in view of their functions
described above. Anti-CD40 antibodies with superior antagonistic
activities are described in WO 02/088186. The representative example of
those antagonistic antibodies is 4D11 antibody. The hybridoma 4D11
producing the 4D11 antibody was submitted on 27, Sep. 2001 for
international deposit under the Budapest Treaty, to International Patent
Organisms Depositary, National Institute of Advanced Industrial Science
and Technology (central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan). The
accession number is FERM BP-7758.

[0011] Patent Document 1 WO 02/088186

DISCLOSURE OF THE INVENTION

[0012] The object of the present invention is to create mutants from the
potentially therapeutic anti-CD40 antibodies disclosed in WO 02/088186,
which mutants are designed optimally as pharmaceutical agent.

[0013] As a result of extensive and intensive research, the present
inventors have successfully created novel mutants of the agonistic or
antagonistic antibodies, which mutants may have a higher therapeutic
effect against diseases than known anti-CD40 antibodies, and completed
the present invention based thereon. The basic idea on modification of
the anti-CD40 antibodies according to the present invention will be
described in detail below.

[0014] The present specification shall encompass the description in the
specification and/or drawings of JP Patent Publication (Kokai) No.
2003-431408 which is the basis for the priority of the present
application.

[0019]FIG. 2A illustrates binding of the anti-CD40 antibodies to the CD40
mutant;

[0020]FIG. 2B illustrates binding of the anti-CD40 antibodies to the CD40
mutant;

[0021]FIG. 2c illustrates binding of the anti-CD40 antibodies to the CD40
mutant;

[0022]FIG. 3A shows diagrams indicating that the KM341-1-19 antibody
having a P331S mutation is as active as the original KM341-1-19 antibody
with respect to binding to Ramos cells;

[0023]FIG. 3B shows diagrams indicating that the KM341-1-19 antibody
having a P331S mutation is as active as the original KM341-1-19 antibody
with respect to enhancement of CD95 expression of Ramos cells;

[0024]FIG. 4A shows a diagram indicating that the KM341-1-19 antibody
having a P331S mutation has a lower CDC activity via the rabbit
complement;

[0025]FIG. 4B shows a diagram indicating that the G2/4 antibody has a
lower complement activity when the human complement is used;

[0026] FIG. 5A-1 shows diagrams indicating that conversion of the subclass
of the 2105 antibody from IgG2 into different subclasses has no effect on
its binding to Ramos cells;

[0027] FIG. 5A-2 shows diagrams indicating that conversion of the subclass
of the KM341-1-19 antibody from IgG2 into different subclasses has no
effect on its binding to Ramos cells;

[0028] FIG. 5B-1 shows diagrams indicating that conversion of the subclass
of the 2105 antibody from IgG2 into different subclasses lowers an
activity to enhance CD95 expression of Ramos cells;

[0029] FIG. 5B-2 shows diagrams indicating that conversion of the subclass
of the KM341-1-19 antibody from IgG2 into different subclasses lowers an
activity to enhance CD95 expression of Ramos cells;

[0030] FIG. 6A-1 shows diagrams indicating that the binding capacity of
the KM341-1-19 antibodies to Ramos cells is independent of the varying
structure of the hinge region;

[0031] FIG. 6A-2 shows diagrams indicating that the binding capacity of
the 2105 antibodies to Ramos cells is independent of the varying
structure of the hinge region;

[0032] FIG. 6B-1 shows diagrams indicating that the upper and middle
hinges of the hinge region are important for the activity of the
KM341-1-19 antibodies to enhance CD95 expression of Ramos cells;

[0033] FIG. 6B-2 shows diagrams indicating that the upper and middle
hinges of the hinge region are important for the activity of the 2105
antibodies to enhance CD95 expression of Ramos cells;

[0034]FIG. 7A shows diagrams indicating that conversion of the subclass
of the F72 antibody to IgG2 has no effect on its binding to Ramos cells;

[0035]FIG. 7B shows diagrams indicating that conversion of the subclass
of the F72 antibody to IgG2 raises an activity to enhance CD95 expression
of Ramos cells;

[0036]FIG. 8A shows diagrams indicating that conversion of the subclass
of the 4D11 antibody from IgG1 to IgG4 has no effect on its binding to
Ramos cells;

[0037]FIG. 8B shows diagrams indicating that conversion of the subclass
of the 4D11 antibody from IgG1 to IgG4 inhibits enhancement by the
CD40Ligand of CD95 expression of Ramos cells, at the same extent as
otherwise;

[0038]FIG. 9 shows a diagram indicating that conversion of the subclass
of the 4D11 antibody from IgG1 to IgG4 or IgG4PE lowers the ADCC
activity;

[0039]FIG. 10 shows a diagram indicating that conversion of the subclass
of the 4D11 antibody from IgG1 to IgG4P lowers the CDC activity;

[0040] FIG. 11 illustrates a variation in number of B cells in the blood
(B220-positive cells among the peripheral blood lymphocytes) over time
after 4D11G1, 4D11G4P or 4D11G4PE was administered into human
CD40-transgenic mice;

[0041]FIG. 12A illustrates a higher expression of CD23 of splenic B cells
(CD23-positive cells among the splenic B cells) after each anti-CD40
antibody was administered into human CD40-transgenic mice;

[0042]FIG. 12B illustrates a higher expression of CD86 of splenic B cells
(CD86-positive cells among the splenic B cells) after each anti-CD40
antibody was administered into human CD40-transgenic mice;

[0043]FIG. 12c illustrates a higher expression of CD95 of splenic B cells
(CD95-positive cells among the splenic B cells) after each anti-CD40
antibody was administered into human CD40-transgenic mice;

[0044]FIG. 13A illustrates the suppressive activity of the
antigen-specific antibody (IgG1) production by 4D11 and 281-1-10 in human
CD40-transgenic mice;

[0045]FIG. 13B illustrates the suppressive activity of the
antigen-specific antibody (IgM) production by 4D11 and 281-1-10 in human
CD40-transgenic mice;

[0046]FIG. 14A illustrates the numbers of B cells in the blood
(B220-positive cells among the peripheral blood lymphocytes) during the
suppression assay of the antigen-specific antibody producing activity;

[0047]FIG. 14B illustrates the numbers of splenic B cells (B220-positive
cells among the splenic lymphocytes) during the suppression assay of the
antigen-specific antibody producing activity;

[0048]FIG. 15 illustrates a variation in number of B cells in the blood
(B220-positive cells among the peripheral blood lymphocytes) over time
after 4D11G4P or 4D11G4PE was administered at a dose of 30 mg/kg into
cynomolgus monkeys;

[0057]FIG. 23 illustrates the volume change of the tumor over time from
cell implantation, in a case where 341G2Ser was administered to tumor
bearing mice with Ramos cells implanted therein;

[0058]FIG. 24 illustrates the volume change of the tumor over time from
cell implantation, in a case where 341G2Ser was administered to tumor
bearing mice with T24 cells implanted therein;

[0059]FIG. 25 illustrates the volume change of the tumor over time from
cell implantation, in a case where 341G2Ser was administered to tumor
bearing mice with Hs 766T cells implanted therein; and

[0060]FIG. 26 illustrates the volume change of the tumor over time from
cell implantation, in a case where 341G2Ser was administered to tumor
bearing mice with Capan-2 cells implanted therein.

BEST MODE FOR CARRYING OUT THE INVENTION

1. Modification of Agonistic Antibodies

[0061] Antibodies are essentially molecules that function to protect
living bodies against foreign bodies, such as microorganisms and viruses,
and cancer, and hence they can kill and eliminate such cells binding to
themselves. The lethal activity is composed of two different activities,
called Antibody-Dependent Cellular Cytotoxicity (abbreviated as ADCC
hereinafter) and Complement-Dependent Cytotoxicity (abbreviated as CDC
hereinafter).

[0062] ADCC refers to a type of cytotoxicity induced by activation of
macrophages, NK cells, neutrophil cells, etc., which recognize target
cells by binding to the constant region of the antibody via Fc receptors
expressed on their surface. In contrast, CDC refers to a type of
cytotoxicity induced by activation of a complement system which occurs
through binding of an antibody to an antigen. These activities are known
to vary depending on a subclass of the antibody, which has been found to
be due to a structural difference in the constant region of antibodies
(Charles A. Janeway et al., Immunology, 1997, Current Biology
Ltd./Garland Publishing Inc.).

[0063] Anti-CD40 agonistic antibodies will be more preferable as
therapeutic agent if they have not the activities of ADCC and/or CDC
which may induce cell death of CD40-expressing cells, in terms of
mechanism of immunoactive action. If CD40-expressing cells are injured by
ADCC and/or CDC activities, immunosuppression may occur rather than
desired immunoactivation, resulting in exacerbation of the disease. In
addition, patients suffering from infectious diseases may have higher
ADCC and/or CDC activities. Therefore, when such antibodies are applied
to infectious diseases, it is necessary to evaluate them for safety more
carefully, for example, using more active rabbit complements than those
present in healthy human serum or peripheral blood which could not be
effective to detect the above activities in this situation. Accordingly,
mutants and recombinants were created which had no activity of ADCC or
CDC and examined for their activity.

[0064] Since ADCC and/or CDC activities are known to vary depending on a
subclass of the antibody of interest, conversion of the subclass may
reduce ADCC and/or CDC activities. For the human IgG subclasses, for
example, IgG4 is generally known to be a subclass with low activities of
both ADCC and CDC, and it is reported that IgG2 is CDC active but poorly
active in ADCC, while IgG1 is highly active in both ADCC and CDC (Charles
A. Janeway et al., Immunology, 1997, Current Biology Ltd./Garland
Publishing Inc.). Selection of a particular subclass by taking advantage
of the above characteristics may create a less cytotoxic antibody from
the original antibody. A combination of a specific subclass of antibody
with such a point mutation as described below may create an antibody with
a desired activity. Further, reduction in ADCC and/or CDC activities of
an antibody is reported to be attained by incorporation of a mutation
into its constant region. For instance, L235, D265, D270, K322, P331, and
P329 (each alphabetical letter denotes an amino acid by the single-letter
notation, and each number denotes an EU index proposed by Kabat et al.
(Kabat et. al., Sequences of proteins of Immunological Interest, 1991
Fifth edition); such symbols will be used hereinafter) may play an
important role in complement activation by human IgG, and substitution of
one of those sites by another amino acid may reduce the CDC activity.
Esohe E. Idusogie et. al. J. Immunol. 2000, 164:4178-4184, Yuanyuan Xu
et. al. J. Biol. Chem. 1994, 269:3469-3474, Brekke, O. H. et. al. Eur. J.
Immunol. 1994, 24:2542, Morgan, A., et. al., Immunology 1995, 86:319,
Lund, J., et. al., J. Immunol., 1996, 157:4963, Tao, M. H., et. al., J.
Exp. Med. 1993, 178:661). Specifically, substitution of D270, K322, P329,
or P331 by A may reduce the CDC activity. Substitution of P331 by S or G
may also induce the same thing.

[0067] The present invention has revealed that in some anti-CD40
antibodies, the hinge region of IgG2 is important in expression of their
strong agonistic activities. Replacement of the variable region or the
constant region except the hinge region by a counterpart of any different
subclass, or incorporation of a point mutation thereinto is expected to
not only modulate the ADCC and/or CDC activities, but increase the
productivity of the antibody, its stability during purification and
storage, and its blood kinetics.

[0068] To produce an antibody drug, the stability of the antibody during
purification and storage is very important. Since the antibodies so far
developed belong mainly to the IgG1 subclass, conversion of the variable
region or the constant region except the hinge region to a sequence
derived from the IgG1 subclass will be also effective to improve the
physical properties of the anti-CD40 agonistic antibodies described
above.

[1] A heavy chain of a monoclonal antibody having an agonistic activity,
which binds to CD40, wherein the heavy chain comprises an upper hinge and
a middle hinge derived from a human IgG2, and a constant region with at
least one amino acid deleted or substituted, or with at least one amino
acid added thereto, said deletion, substitution or addition being capable
of increasing or decreasing ADCC and/or CDC. [2] The heavy chain
according to [1], wherein the constant region is derived from a human
IgG. [3] The heavy chain according to [2], wherein the human IgG is a
human IgG1. [4] The heavy chain according to [2], wherein the human IgG
is a human IgG2. [5] The heavy chain according to [2], wherein the human
IgG is a human IgG3. [6] The heavy chain according to [2], wherein the
human IgG is a human IgG4. [7] The heavy chain according to any of [3] to
[5], wherein said substitution of amino acids in the constant region is
substitution of proline with serine at position 331 which is indicated by
the EU index as in Kabat et al. [8] A monoclonal antibody comprising the
heavy chain according to any of [1] to [7]. [9] The heavy chain according
to any of [1] to [7], wherein the heavy chain comprises a variable region
from a heavy chain of a monoclonal antibody produced by the hybridoma
KM341-1-19 (Accession No. FERM BP-7759). [10] A monoclonal antibody
consisting of the heavy chain according to [9] and a light chain
comprising a variable region from a light chain of a monoclonal antibody
produced by the hybridoma KM341-1-19 (Accession No. FERM BP-7759). [11]
The heavy chain according to any of [1] to [7], wherein the heavy chain
comprises a variable region of the polypeptide represented by SEQ ID NO:
38. [12] A monoclonal antibody consisting of the heavy chain according to
[11] and a light chain of a monoclonal antibody, wherein the light chain
comprises a variable region of the polypeptide represented by SEQ ID NO:
40. [13] The heavy chain according to [1], wherein the heavy chain
consists of a remaining portion provided by removing the signal sequence
from the polypeptide represented by SEQ ID NO: 132. [14] A monoclonal
antibody consisting of the heavy chain according to [13] and a light
chain of a monoclonal antibody, wherein the light chain consists of a
remaining portion provided by removing the signal sequence from the
polypeptide represented by SEQ ID NO: 134. [15] The heavy chain according
to [1], wherein the heavy chain is produced by a host comprising an
expression vector having the polynucleotide represented by SEQ ID NO:
131. [16] The monoclonal antibody according to [8], wherein the
monoclonal antibody is produced by a host comprising an expression vector
having the polynucleotide represented by SEQ ID NO: 131 and the
polynucleotide represented by SEQ ID NO: 133. [17] The heavy chain
according to any of [1] to [7], wherein the heavy chain comprises a
variable region from a heavy chain of a monoclonal antibody produced by
the hybridoma 2105 (Accession No. FERM BP-8024). [18] A monoclonal
antibody consisting of the heavy chain according to [17] and a light
chain comprising a variable region from a light chain of a monoclonal
antibody produced by the hybridoma 2105 (Accession No. FERM BP-8024).
[19] The heavy chain according to any of [1] to [7], wherein the heavy
chain comprises a variable region of the polypeptide represented by SEQ
ID NO: 42. [20] A monoclonal antibody consisting of the heavy chain
according to [19] and a light chain of a monoclonal antibody, wherein the
light chain comprises a variable region of the polypeptide represented by
SEQ ID NO: 44. [21] The heavy chain according to [1], wherein the heavy
chain consists of a remaining portion provided by removing the signal
sequence from the polypeptide represented by SEQ ID NO: 136. [22] A
monoclonal antibody consisting of the heavy chain according to [21] and a
light chain of a monoclonal antibody, wherein the light chain consists of
a remaining portion provided by removing the signal sequence from the
polypeptide represented by SEQ ID NO: 138. [23] The heavy chain according
to [1], wherein the heavy chain is produced by a host comprising an
expression vector having the polynucleotide represented by SEQ ID NO:
135. [24] The monoclonal antibody according to [8], wherein the
monoclonal antibody is produced by a host comprising an expression vector
having the polynucleotide represented by SEQ ID NO: 135 and the
polynucleotide represented by SEQ ID NO: 137. [25] A polynucleotide
represented by SEQ ID NO: 131. [26] A polynucleotide represented by SEQ
ID NO: 133. [27] An expression vector having the polynucleotide according
to [25]. [28] An expression vector having the polynucleotide according to
[26]. [29] An expression vector having the polynucleotides according to
[25] and [26]. [30] A host comprising the expression vector according to
[27]. [31] A host comprising the expression vector according to [28].
[32] A host comprising the expression vector according to [29]. [33] A
process of producing a heavy chain of a monoclonal antibody, comprising
the steps of: culturing the host according to [30] in a culture medium;
and obtaining a heavy chain of a monoclonal antibody from the culture
and/or the host. [34] A process of producing a monoclonal antibody,
comprising the steps of: culturing the host according to [32] in a
culture medium; and obtaining a monoclonal antibody from the culture
and/or the host. [35] A polynucleotide represented by SEQ ID NO: 135.
[36] A polynucleotide represented by SEQ ID NO: 137. [37] An expression
vector having the polynucleotide according to [35]. [38] An expression
vector having the polynucleotide according to [36]. [39] An expression
vector having the polynucleotides according to [35] and [36]. [40] A host
comprising the expression vector according to [37]. [41] A host
comprising the expression vector according to [38]. [42] A host
comprising the expression vector according to [39]. [43] A process of
producing a heavy chain of a monoclonal antibody, comprising the steps
of: culturing the host according to [40] in a culture medium; and
obtaining a heavy chain of a monoclonal antibody from the culture and/or
the host. [44] A process of producing a a monoclonal antibody, comprising
the steps of: culturing the host according to [42] in a culture medium;
and obtaining a monoclonal antibody from the culture and/or the host.
[45] A process of producing a heavy chain of a monoclonal antibody having
an agonistic activity capable of binding to CD40, comprising the step of
substituting the upper hinge and the middle hinge of an antibody, which
is not either an upper hinge or a middle hinge derived from a human IgG2,
with an upper hinge and a middle hinge derived from a human IgG2,
respectively. [46] A process of producing a heavy chain of a monoclonal
antibody comprising a variable region, and an upper hinge and a middle
hinge derived from a human IgG2, comprising the step of identifying a
polypeptide forming the variable region, which is from a heavy chain of a
monoclonal antibody capable of binding to CD40. [47] A process of
producing a monoclonal antibody having an agonistic activity capable of
binding to CD40, comprising the step of substituting the upper hinge and
the middle hinge of an antibody, which is not either an upper hinge or a
middle hinge derived from a human IgG2, with an upper hinge and a middle
hinge derived from a human IgG2, respectively. [48] A process of
producing a monoclonal antibody comprising a variable region, and an
upper hinge and a middle hinge derived from a human IgG2, comprising the
step of identifying a polypeptide forming the variable region, which is
from a heavy chain of a monoclonal antibody capable of binding to CD40.
[49] A pharmaceutical composition comprising the monoclonal antibody
according to [8], [10], [12], [14], [16], [18], [20], [22] or [24] as an
active ingredient. [50] The pharmaceutical composition according to [49]
used for prevention or treatment of a malignant tumor, a pathogen or an
autoimmune disease. [51] A method of prevention or treatment of a
malignant tumor, a pathogen or an autoimmune disease, comprising
administration of the pharmaceutical composition according to [49] into a
mammal. [52] Use of the monoclonal antibody according to [8], [10], [12],
[14], [16], [18], [20], [22] or [24] for production of a pharmaceutical
composition used for prevention or treatment of a malignant tumor, a
pathogen or an autoimmune disease. [89] A polynucleotide provided by
removing the portion encoding the signal sequence from the polynucleotide
represented by SEQ ID NO: 131. [90] A polynucleotide provided by removing
the portion encoding the signal sequence from the polynucleotide
represented by SEQ ID NO: 133. [91] A polynucleotide provided by removing
the portion encoding the signal sequence from the polynucleotide
represented by SEQ ID NO: 135. [92] A polynucleotide provided by removing
the portion encoding the signal sequence from the polynucleotide
represented by SEQ ID NO: 137.

[0070] The present invention provides an antibody produced by modification
of an agonistic anti-CD40 antibody belonging to the human IgG2, wherein
the modified antibody is a mutant having the constant region, exclusive
of the upper and middle hinges, substituted with a sequence derived from
a different subclass. The subclass is preferably IgG1. The present
invention provides an antibody produced by modification of an agonistic
anti-CD40 antibody belonging to the human IgG2, wherein the modified
antibody is a mutant having the constant region, exclusive of the hinge
region, substituted with a sequence derived from a different subclass.
The subclass is preferably IgG1.

[0071] Herein, reduction in ADCC and CDC activities means reduction in
those activities as compared with the corresponding activities of an
anti-CD40 monoclonal antibody other than the mutants described above, for
example, as compared with the corresponding activities of a monoclonal
antibody produced by the hybridoma KM341-1-19 (Accession No. FERM
BP-7759) or 2105 (Accession No. FERM BP-8024). The ADCC and CDC
activities may be assayed by any known method, for example, the method
described in the Examples herein. The sequences of variable regions in
the heavy and light chains of a monoclonal antibody will be presented
below which is produced by the hybridoma KM341-1-19 (Accession No. FERM
BP-7759) or 2105 (Accession No. FERM BP-8024).

[0072] DNA encoding variable regions in the heavy and light chains of the
KM341-1-19 antibody and the amino acid sequences of the heavy and light
chains will be presented below.

[0073] In the heavy chain nucleotide sequence (SEQ ID NO: 37) of the
KM341-1-19 antibody, the signal sequence is initiated with adenine (A) at
position 50. The boundary between the signal sequence and the variable
region is located between "adenine" ([A]) at position 109 and cytosine
(C) at position 110, and the boundary between the variable region and the
constant region is located between adenine (A) at position 493 and
guanine (G) at position 494 (the gene prediction software (Signal P ver.
2) was used).

[0074] In the heavy chain amino acid sequence (SEQ ID NO: 38) of the
KM341-1-19 antibody, the boundary between the signal sequence and the
variable region is located between serine (S) at position 20 and
glutamine (Q) at position 21, and the boundary between the variable
region and the constant region is located between serine (S) at position
148 and alanine (A) at position 149.

[0075] Accordingly, the variable region in the heavy chain of the
KM341-1-19 antibody has a nucleotide sequence ranging from cytosine (C)
at position 110 to adenine (A) at position 493, as seen in SEQ ID NO: 37.
Further, the variable region in the heavy chain of the KM341-1-19
antibody has an amino acid sequence ranging from glutamine (Q) at
position 21 to serine (S) at position 148, as seen in SEQ ID NO: 38.

[0076] In the light chain nucleotide sequence (SEQ ID NO: 39) of the
KM341-1-19 antibody, the signal sequence is initiated with adenine (A) at
position 29. The boundary between the signal sequence and the variable
region is located between "adenine" ([A]) at position 88 and guanine (G)
at position 89, and the boundary between the variable region and the
constant region is located between adenine (A) at position 400 and
"cytosine" ([C]) at position 401 (the gene prediction software (Signal P
ver. 2) was used).

[0077] In the light chain amino acid sequence (SEQ ID NO: 40) of the
KM341-1-19 antibody, the boundary between the signal sequence and the
variable region is located between glycine (G) at position 20 and
glutamic acid (E) at position 21, and the boundary between the variable
region and the constant region is located between lysine (K) at position
124 and "arginine" ([R]) at position 125.

[0078] Accordingly, the variable region in the light chain of the
KM341-1-19 antibody has a nucleotide sequence ranging from guanine (G) at
position 89 to adenine (A) at position 400, as seen in SEQ ID NO: 39.
Further, the variable region in the light chain of the KM341-1-19
antibody has an amino acid sequence ranging from glutamic acid (E) at
position 21 to lysine (K) at position 124, as seen in SEQ ID NO: 40.

[0079] DNA encoding variable regions in the heavy and light chains of the
2105 antibody and the amino acid sequences of the heavy and light chains
will be presented below.

[0080] In the heavy chain nucleotide sequence (SEQ ID NO: 41) of the 2105
antibody, the signal sequence is initiated with adenine (A) at position
70. The boundary between the signal sequence and the variable region is
located between "thymine" ([T]) at position 126 and guanine (G) at
position 127, and the boundary between the variable region and the
constant region is located between adenine (A) at position 495 and
guanine (G) at position 496 (the gene prediction software (Signal P ver.
2) was used).

[0081] In the heavy chain amino acid sequence (SEQ ID NO: 42) of the 2105
antibody, the boundary between the signal sequence and the variable
region is located between cysteine (C) at position 19 and glutamic acid
(E) at position 20, and the boundary between the variable region and the
constant region is located between serine (S) at position 142 and alanine
(A) at position 143.

[0082] Accordingly, the variable region in the heavy chain of the 2105
antibody has a nucleotide sequence ranging from guanine (G) at position
127 to adenine (A) at position 495, as seen in SEQ ID NO: 41. Further,
the variable region in the heavy chain of the 2105 antibody has an amino
acid sequence ranging from glutamic acid (E) at position 20 to serine (S)
at position 142, as seen in SEQ ID NO: 42.

[0083] In the light chain nucleotide sequence (SEQ ID NO: 43) of the 2105
antibody, the signal sequence is initiated with adenine (A) at position
28. The boundary between the signal sequence and the variable region is
located between "adenine" ([A]) at position 87 and guanine (G) at
position 88, and the boundary between the variable region and the
constant region is located between adenine (A) at position 405 and
"cytosine" ([C]) at position 406 (the gene prediction software (Signal P
ver. 2) was used).

[0084] In the light chain amino acid sequence (SEQ ID NO: 44) of the 2105
antibody, the boundary between the signal sequence and the variable
region is located between glycine (G) at position 20 and glutamic acid
(E) at position 21, and the boundary between the variable region and the
constant region is located between lysine (K) at position 126 and
"arginine" ([R]) at position 127.

[0085] Accordingly, the variable region in the light chain of the 2105
antibody has a nucleotide sequence ranging from guanine (G) at position
88 to adenine (A) at position 405, as seen in SEQ ID NO: 43. Further, the
variable region in the light chain of the 2105 antibody has an amino acid
sequence ranging from glutamic acid (E) at position 21 to lysine (K) at
position 126, as seen in SEQ ID NO: 44.

[0086] In the heavy chain nucleotide sequence (SEQ ID NO: 131) of the
341G2Ser, the boundary between the signal sequence and the variable
region is located between "adenine" ([A]) at position 60 and cytosine (C)
at position 61, and the boundary between the variable region and the
constant region is located between adenine (A) at position 444 and
guanine (G) at position 445 (the gene prediction software (Signal P ver.
2) was used).

[0087] In the heavy chain amino acid sequence (SEQ ID NO: 132) of the
341G2Ser, the boundary between the signal sequence and the variable
region is located between serine (S) at position 20 and glutamine (Q) at
position 21, and the boundary between the variable region and the
constant region is located between serine (S) at position 148 and alanine
(A) at position 149.

[0088] Accordingly, the variable region in the heavy chain of the 341G2Ser
has a nucleotide sequence ranging from cytosine (C) at position 61 to
adenine (A) at position 444, as seen in SEQ ID NO: 131. Further, the
variable region in the heavy chain of the 341G2Ser has an amino acid
sequence ranging from glutamine (Q) at position 21 to serine (S) at
position 148, as seen in SEQ ID NO: 132.

[0089] In the light chain nucleotide sequence (SEQ ID NO: 133) of the
341G2Ser, the boundary between the signal sequence and the variable
region is located between "adenine" ([A]) at position 60 and guanine (G)
at position 61, and the boundary between the variable region and the
constant region is located between adenine (A) at position 372 and
"cytosine" ([C]) at position 373 (the gene prediction software (Signal P
ver. 2) was used).

[0090] In the light chain amino acid sequence (SEQ ID NO: 134) of the
341G2Ser, the boundary between the signal sequence and the variable
region is located between glycine (G) at position 20 and glutamic acid
(E) at position 21, and the boundary between the variable region and the
constant region is located between lysine (K) at position 124 and
"arginine" ([R]) at position 125. Accordingly, the variable region in the
light chain of the 341G2Ser has a nucleotide sequence ranging from
guanine (G) at position 61 to adenine (A) at position 372, as seen in SEQ
ID NO: 133. Further, the variable region in the light chain of the
341G2Ser has an amino acid sequence ranging from glutamic acid (E) at
position 21 to lysine (K) at position 124, as seen in SEQ ID NO: 134.

[0091] In the heavy chain nucleotide sequence (SEQ ID NO: 135) of the
2105G2Ser, the boundary between the signal sequence and the variable
region is located between "thymine" ([T]) at position 57 and guanine (G)
at position 58, and the boundary between the variable region and the
constant region is located between adenine (A) at position 426 and
guanine (G) at position 427 (the gene prediction software (Signal P ver.
2) was used).

[0092] In the heavy chain amino acid sequence (SEQ ID NO: 136) of the
2105G2Ser, the boundary between the signal sequence and the variable
region is located between cysteine (C) at position 19 and glutamic acid
(E) at position 20, and the boundary between the variable region and the
constant region is located between serine (S) at position 142 and alanine
(A) at position 143.

[0093] Accordingly, the variable region in the heavy chain of the
2105G2Ser has a nucleotide sequence ranging from guanine (G) at position
58 to adenine (A) at position 426, as seen in SEQ ID NO: 135. Further,
the variable region in the heavy chain of the 2105G2Ser has an amino acid
sequence ranging from glutamic acid (E) at position 20 to serine (S) at
position 142, as seen in SEQ ID NO: 136.

[0094] In the light chain nucleotide sequence (SEQ ID NO: 137) of the
2105G2Ser, the boundary between the signal sequence and the variable
region is located between "adenine" ([A]) at position 60 and guanine (G)
at position 61, and the boundary between the variable region and the
constant region is located between adenine (A) at position 378 and
"cytosine" ([C]) at position 379 (the gene prediction software (Signal P
ver. 2) was used).

[0095] In the light chain amino acid sequence (SEQ ID NO: 138) of the
2105G2Ser, the boundary between the signal sequence and the variable
region is located between glycine (G) at position 20 and glutamic acid
(E) at position 21, and the boundary between the variable region and the
constant region is located between lysine (K) at position 126 and
"arginine" ([R]) at position 127. Accordingly, the variable region in the
light chain of the 2105G2Ser has a nucleotide sequence ranging from
guanine (G) at position 61 to adenine (A) at position 378, as seen in SEQ
ID NO: 137. Further, the variable region in the light chain of the
2105G2Ser has an amino acid sequence ranging from glutamic acid (E) at
position 21 to lysine (K) at position 126, as seen in SEQ ID NO: 138.

[0096] Anti-CD40 antagonistic antibodies will be more preferable as
therapeutic agent as well as the agonistic antibodies, if they have not
the activities of ADCC and/or CDC, in terms of mechanism of action.
Furthermore, it is important that anti-CD40 antagonistic antibodies have
no activity to induce signals by their in vivo crosslinking via Fc
receptors, even if the ADCC activity cannot be detected. In other words,
it is necessary to confirm that they are not activate the immunity, and
such active antibodies may be desired as pharmaceutical agent. Anti-CD40
antagonistic antibodies are promising as therapeutic agent for treating
autoimmune diseases or suppressing rejection in organ transplantation. If
they induce an agonistic activity due to some effect after they are
administered to patients, however weak it may be, the symptoms may worsen
in contrast to the desired therapeutic effect. Thus, an antibody without
any agonistic activity is more preferable as pharmaceutical agent. In the
present invention, incorporation of a point mutation L235E (means
substitution of L at position 235 with E; similar symbols will be used
hereinafter) into IgG4 has been demonstrated to be effective for in vivo
reduction in the agonistic activity, in the animal test using monkeys.
Although IgG4 is a subclass with low activities of ADCC and CDC, it is
reported that when it was attempted to express IgG4 as recombinant
protein in cells like CHO, its half-molecules were secreted due to a poor
S--S bonding between the heavy chains (Rob C. Aalberse et al.,
Immunology, 105, 9-19, 2002). To overcome this problem, incorporation of
a mutation into the constant region of antibodies is reported to
successfully promote the formation of the S--S bonding. Therefore, this
type of mutation was also evaluated for its usefulness. Specifically, the
mutation of substituting S at position 228 with P was incorporated (S.
Angal et al., Molecular Immunology, vol. 30, no. 1, 105-108, 1993).

[0097] For antagonistic antibodies as well as agonistic antibodies, the
stability of the antibody during purification and storage is very
important. There may be some methods to create such antibodies as are
physically better while keeping the antagonistic activity. The antibody
pharmaceuticals so far offered commercially belong mostly to the IgG1
subclass, and they are not reported to be problematic in pharmaceutical
formulation. Based on these facts, it may be advantageous from the
viewpoint of physical properties to derive the constant region of
antibodies from IgG1. In case of anti-CD40 antibodies, however, they are
desirably lower in ADCC and CDC activities. As a consequence, antibodies
having an IgG1-type constant region modified with some point mutations
may be desired. The mutations described above are useful to create such
antibodies. The IgG1-type constant region may become lower in ADCC and
CDC activities by incorporating point mutation P331G thereinto. It is
also observed that incorporation of point mutation L235E into IgG4
eliminates a slight agonistic activity in vivo to make it
pharmaceutically more active, but makes it physically less stable at a
low pH. Thus, substitution of L235 with an amino acid other than E may
make it physically more functional. As to the 4D11 antibody, it is very
similar to the 2B 11 antibody with respect to the structure of its
variable region. The 2B11 antibody has a lower antagonistic activity, but
has a higher stability at a low pH, compared with the 4D11 antibody. If
some amino acids derived from the constant region of 2B11 are
incorporated into the 4D11 antibody, based on the above properties, 4D11
may become more stable. Specifically, point mutation L38V, P58R, G62W,
I79M, K81Y, H87Y, S98A, K109R, V120M or T124A in the heavy chain, or N75S
in the light chain, or a combination thereof may be effective for that
purpose. Specifically, a mutant created by substituting L at position 38
in the variable region of the heavy chain of the 4D11 antibody with V
(abbreviated as L38V; similar symbols will be used hereinafter), a P58R
mutant, a G62W mutant, a I79M mutant, a K81Y mutant, a H87Y mutant, a
S98A mutant, a K109R mutant, a V120M mutant or a T124A mutant, or a N75S
mutant in the light chain, or a combination thereof may be provided for
that purpose.

[53] A heavy chain of a monoclonal antibody having an antagonistic
activity capable of binding to CD40, wherein the heavy chain comprises a
constant region with at least one amino acid deleted or substituted, or
with at least one amino acid added thereto, said deletion, substitution
or addition being capable of increasing or decreasing ADCC and/or CDC.
[54] The heavy chain according to [53], wherein the constant region is
derived from a human IgG. [55] The heavy chain according to [54], wherein
the human IgG is a human IgG1. [56] The heavy chain according to [54],
wherein the human IgG is a human IgG2. [57] The heavy chain according to
[54], wherein the human IgG is a human IgG3. [58] The heavy chain
according to [54], wherein the human IgG is a human IgG4. [59] The heavy
chain according to any of [55], [57] or [58], wherein said substitution
of amino acids in the constant region is substitution of leucine with
glutamic acid at position 235 which is indicated by the EU index as in
Kabat et al. [60] A heavy chain according to any of [53] to [58], wherein
the heavy chain comprises a constant region with at least one amino acid
deleted or substituted, or with at least one amino acid added thereto,
said deletion, substitution or addition being capable of promoting the
formation of the S--S bond between the heavy chains. [61] The antibody
heavy chain according to [60], wherein said substitution of amino acids
in the constant region is substitution of serine with proline at position
228 which is indicated by the EU index as in Kabat et al. [62] A
monoclonal antibody comprising the heavy chain according to any of [53]
to [61]. [63] The heavy chain according to any of [53] to [61], wherein
the heavy chain comprises a variable region from a heavy chain of a
monoclonal antibody produced by the hybridoma 4D11 (Accession No. FERM
BP-7758). [64] A monoclonal antibody comprising the heavy chain according
to [63] and a light chain comprising a variable region from a light chain
of a monoclonal antibody produced by the hybridoma 4D11 (Accession No.
FERM BP-7758). [65] The heavy chain according to any of [53] to [61],
wherein the heavy chain comprises a variable region of the polypeptide
represented by SEQ ID NO: 46. [66] A monoclonal antibody consisting of
the heavy chain according to [65] and a light chain of a monoclonal
antibody, wherein the light chain comprises a variable region of the
polypeptide represented by SEQ ID NO: 48. [67] The heavy chain according
to [53], wherein the heavy chain consists of a remaining portion provided
by removing the signal sequence from the polypeptide represented by SEQ
ID NO: 140. [68] A monoclonal antibody consisting of the heavy chain
according to [67] and a light chain of a monoclonal antibody, wherein the
light chain consists of a remaining portion provided by removing the
signal sequence from the polypeptide represented by SEQ ID NO: 142. [69]
The heavy chain according to [53], wherein the heavy chain is produced by
a host comprising an expression vector having the polynucleotide
represented by SEQ ID NO: 139. [70] The monoclonal antibody according to
[62], wherein the monoclonal antibody is produced by a host comprising an
expression vector having the polynucleotide represented by SEQ ID NO: 139
and the polynucleotide represented by SEQ ID NO: 141. [71] A
polynucleotide represented by SEQ ID NO: 139. [72] A polynucleotide
represented by SEQ ID NO: 141. [73] An expression vector having the
polynucleotide according to [71]. [74] An expression vector having the
polynucleotide according to [72]. [75] An expression vector having the
polynucleotides according to [71] and [72]. [76] A host comprising the
expression vector according to [73]. [77] A host comprising the
expression vector according to [74]. [78] A host comprising the
expression vector according to [75]. [79] A process of producing a heavy
chain of a monoclonal antibody, comprising the steps of: culturing the
host according to [76] in a culture medium; and obtaining a heavy chain
of a monoclonal antibody from the culture and/or the host. [80] A process
of producing a monoclonal antibody, comprising the steps of: culturing
the host according to [78] in a culture medium; and obtaining a
monoclonal antibody from the culture and/or the host. [81] A
pharmaceutical composition comprising the monoclonal antibody according
to [62], [64], [66], [68] or [70] as an active ingredient. [82] The
pharmaceutical composition according to [81] used for prevention or
treatment of transplant rejection, an autoimmune disease, allergy or
blood clotting factor VIII inhibition. [83] A method of prevention or
treatment of transplant rejection, an autoimmune disease, allergy or
blood clotting factor VIII inhibition, which comprises administering the
pharmaceutical composition according to [81] into a mammal. [84] Use of
the monoclonal antibody according to [62], [64], [66], [68] or [70] for
production of a pharmaceutical composition used for prevention or
treatment of transplant rejection, an autoimmune disease, allergy or
blood clotting factor VIII inhibition. [85] A method of producing a heavy
chain of a monoclonal antibody having an antagonistic activity capable of
binding to CD40, wherein the agonistic activity is lowered, comprising a
step of making deletion or substitution of at least one amino acid, or
addition of at least one amino acid in a constant region of a heavy chain
of a human antibody. [86] The method according to [85], wherein the
constant region is from a human IgG. [87] The method according to [86],
wherein the human IgG is a human IgG4. [88] The method according to any
of [85] to [87], wherein said substitution of amino acids in the constant
region is substitution of leucine with glutamic acid at position 235
which is indicated by the EU index as in Kabat et al. [93] A
polynucleotide provided by removing the portion encoding the signal
sequence from the polynucleotide represented by SEQ ID NO: 139. [94] A
polynucleotide provided by removing the portion encoding the signal
sequence from the polynucleotide represented by SEQ ID NO: 141.

[0101] A mutant of an antagonistic anti-CD40 antibody, comprising at least
one substitution selected from the group consisting of substitution of L
with V at position 38, substitution of P with R at position 58,
substitution of G with W at position 62, substitution of I with M at
position 79, substitution of K with Y at position 81, substitution of H
with Y at position 87, substitution of S with A at position 98,
substitution of K with Rat position 109, substitution of V with M at
position 120 and substitution of T with A at position 124, which
substitutions are all carried out in a variable region of a heavy chain
of a monoclonal antibody produced by the hybridoma 4D11 (Accession No.
FERM BP-7758), and a mutant of an antagonistic anti-CD40 antibody
comprising substitution of N with S at position 75 in a variable region
of a light chain of the 4D11 antibody.

[0102] Herein, reduction in ADCC and CDC activities means reduction in
those activities as compared with the corresponding activities of an
anti-CD40 monoclonal antibody other than the mutants described above, for
example, as compared with the corresponding activities of a monoclonal
antibody produced by the hybridoma 4D11 (Accession No. FERM BP-7758). The
ADCC and CDC activities may be assayed by any known method, for example,
the method described in the Examples herein. The sequences of variable
regions in the heavy and light chains of a monoclonal antibody will be
presented below which is produced by the hybridoma 4D11 (Accession No.
FERM BP-7758).

[0103] DNA encoding variable regions in the heavy and light chains of the
4D11 antibody and the amino acid sequences of the heavy and light chains
will be presented below, respectively.

[0104] In the heavy chain nucleotide sequence (SEQ ID NO: 45) of the 4D11
antibody, the boundary between the signal sequence and the variable
region is located between "cytosine" ([C]) at position 93 and cytosine
(C) at position 94, and the boundary between the variable region and the
constant region is located between adenine (A) at position 456 and
guanine (G) at position 457 (the gene prediction software (Signal P ver.
2) was used).

[0105] In the heavy chain amino acid sequence (SEQ ID NO: 46) of the 4D11
antibody, the boundary between the signal sequence and the variable
region is located between serine (S) at position 26 and glutamine (Q) at
position 27, and the boundary between the variable region and the
constant region is located between serine (S) at position 147 and alanine
(A) at position 148.

[0106] Accordingly, the variable region in the heavy chain of the 4D11
antibody has a nucleotide sequence ranging from cytosine (C) at position
94 to adenine (A) at position 456, as seen in SEQ ID NO: 45. Further, the
variable region in the heavy chain of the 4D11 antibody has an amino acid
sequence ranging from glutamine (Q) at position 27 to serine (S) at
position 147, as seen in SEQ ID NO: 46.

[0107] In the light chain nucleotide sequence (SEQ ID NO: 47) of the 4D11
antibody, the boundary between the signal sequence and the variable
region is located between "thymine" ([T]) at position 124 and guanine (G)
at position 125, and the boundary between the variable region and the
constant region is located between adenine (A) at position 442 and
"cytosine" ([C]) at position 443 (the gene prediction software (Signal P
ver. 2) was used).

[0108] In the light chain amino acid sequence (SEQ ID NO: 48) of the 4D11
antibody, the boundary between the signal sequence and the variable
region is located between cytosine (C) at position 22 and alanine (A) at
position 23, and the boundary between the variable region and the
constant region is located between lysine (K) at position 128 and
"arginine" ([R]) at position 129.

[0109] Accordingly, the variable region in the light chain of the 4D11
antibody has a nucleotide sequence ranging from guanine (G) at position
125 to adenine (A) at position 442, as seen in SEQ ID NO: 47. Further,
the variable region in the light chain of the 4D11 antibody has an amino
acid sequence ranging from alanine (A) at position 23 to lysine (K) at
position 128, as seen in SEQ ID NO: 48.

[0110] In the heavy chain nucleotide sequence (SEQ ID NO: 139) of the 4D11
antibody G4PE, the boundary between the signal sequence and the variable
region is located between "cytosine" ([C]) at position 78 and cytosine
(C) at position 79, and the boundary between the variable region and the
constant region is located between adenine (A) at position 441 and
guanine (G) at position 442 (the gene prediction software (Signal P ver.
2) was used).

[0111] In the heavy chain amino acid sequence (SEQ ID NO: 140) of the 4D11
antibody, the boundary between the signal sequence and the variable
region is located between serine (S) at position 26 and glutamine (Q) at
position 27, and the boundary between the variable region and the
constant region is located between serine (S) at position 147 and alanine
(A) at position 148.

[0112] Accordingly, the variable region in the heavy chain of the 4D11
antibody has a nucleotide sequence ranging from cytosine (C) at position
79 to adenine (A) at position 441, as seen in SEQ ID NO: 139. Further,
the variable region in the heavy chain of the 4D11 antibody has an amino
acid sequence ranging from glutamine (Q) at position 27 to serine (S) at
position 147, as seen in SEQ ID NO: 140.

[0113] In the light chain nucleotide sequence (SEQ ID NO: 141) of the
4D11G4PE, the boundary between the signal sequence and the variable
region is located between "thymine" ([T]) at position 66 and guanine (G)
at position 67, and the boundary between the variable region and the
constant region is located between adenine (A) at position 384 and
"cytosine" ([C]) at position 385 (the gene prediction software (Signal P
ver. 2) was used).

[0114] In the light chain amino acid sequence (SEQ ID NO: 142) of the
4D11G4PE, the boundary between the signal sequence and the variable
region is located between cytosine (C) at position 22 and alanine (A) at
position 23, and the boundary between the variable region and the
constant region is located between lysine (K) at position 128 and
"arginine" ([R]) at position 129.

[0115] Accordingly, the variable region in the light chain of the 4D11G4PE
has a nucleotide sequence ranging from guanine (G) at position 67 to
adenine (A) at position 384, as seen in SEQ ID NO: 141. Further, the
variable region in the light chain of the 4D11 antibody has an amino acid
sequence ranging from alanine (A) at position 23 to lysine (K) at
position 128, as seen in SEQ ID NO: 142.

[0118] "An anti-CD40 antibody" refers to any monoclonal antibody to a
cell-expressed CD40, a full-length CD40 or a partial-length CD40.

[0119] In addition, "an antibody" of the invention is derived from genes
(collectively called antibody genes) encoding a heavy chain variable
region and a heavy chain constant region, as well as a light chain
variable region and a light chain constant region which together
constitute an immunoglobulin. Human immunoglobulins are grouped into 5
different classes consisting of IgG, IgA, IgM, IgD, and IgE. Further, IgG
is composed of 4 different subclasses, IgG1, IgG2, IgG3 and IgG4, while
IgA is composed of 2 different subclasses, IgA1 and IgA2. IgG1, IgG2,
IgG3 and IgG4 are located in 14q32, 33 of the human chromosomes. The
fundamental structure of immunoglobulin is composed of two homologous L
chains (light chains) and two homologous H chains (heavy chains). The
class and subclass of an immunoglobulin is determined by its H chains.
The antibody according to the present invention may comprise any class,
any subclass or any isotype of immunoglobulin. "A functional fragment" of
the inventive antibody refers to a portion (partial fragment) of the
antibody defined above that is active singly or multiply on an antigen to
the antibody, including, for example, F(ab')2, Fab', Fab, Fv,
disulfide-stabilized FV, single-chain FV (scFV) and a multimer thereof
(D. J. King, Applications and Engineering of Monoclonal Antibodies, 1998,
T. J. International Ltd.).

[0121] In the present invention, CH1, hinge, CH2 and CH3 each denote a
portion of the heavy-chain constant region of any antibody, and are based
on the EU index as in Kabat et al. (Kabat et al., Sequences of proteins
of immunological interest, 1991 Fifth edition). By definition, CH1 ranges
from 118 to 215 by the EU index, hinge ranges from 216 to 230 by the EU
index, CH2 ranges from 231 to 340 by the EU index, and CH3 ranges from
341 to 446 by the EU index.

[0122] The "human antibody" of the present invention means an antibody
which is an expression product of a human-derived antibody gene.

[0123] "Agonistic" refers to an action of enhancing binding of a ligand to
CD40 expressed on the surface of such cells as B cells, tumor cells or
dendritic cells, or an action of providing the CD40-expressing cells with
at least one effect which the CD40 ligand makes on the CD40-expressing
cells. "An agonistic antibody" refers to an antibody having such an
agonistic action. An example of the effects provided for the
CD40-expressing cells is to promote the expression of CD95.

[0124] "Antagonistic" refers to an action of inhibiting binding of the
ligand to CD40 expressed on the surface of such cells as B cells, tumor
cells or dendritic cells, or an action of neutralizing at least one
effect which the CD40 ligand makes on the CD40-expressing cells. "An
antagonistic antibody" refers to an antibody having such an antagonistic
action. An example of the effects provided for the CD40-expressing cells
is to suppress the proliferation of B cells or the production of
antibodies.

[0127] The anti-CD40 antibody according to the present invention can be
provided by incorporating an antibody gene into an expression vector,
transfecting the vector into a suitable host cell, harvesting the
antibody from the cultured cells or the supernatant, and purifying it.

[0128] The vector may be a phage or a plasmid which can replicate in the
host cell by itself or can be integrated into the chromosome of the host
cell. The plasmid DNA may be derived from Escherichia coli, Bacillus
subtilis or a yeast, while the phage DNA may be from λ phage.

[0129] The host cell for transformation is not particularly limited if it
can express the target gene. Examples of the host cell may include
bacteria (Escherichia coli, Bacillus subtilis, etc.), yeasts, animal
cells (COS cell, CHO cell, etc.) and insect cells.

[0130] There are known modes of transferring the gene into the host cells,
including any mode, such as mediation by calcium ion, electroporation,
spheroplast fusion, mediation by lithium acetate, calcium phosphate
transfection or lipofection. In order to transfer the gene into an
animal, as described later, the modes include microinjection;
electroporation or lipofection for ES cells; and nuclear transplantation.

[0131] In the present invention, "culture" refers to (a) culture
supernatant, (b) cultured cells, cultured biomass or disrupted matter
thereof, or (c) secretion of transformant. To culture the transformant, a
medium suitable for the host is used and static culture, roller bottle
culture or something else may be employed.

[0132] After the culture, if the desired antibody protein is produced
within the biomass or cells, the antibody is harvested by disrupting the
biomass or cells. If the desired antibody is produced out of the biomass
or cells, the culture solution is used as it is or after it is separated
from the biomass or cells by centrifugation or other means. Thereafter, a
biochemical process utilizing any chromatography, which is appropriate
for separation/purification of proteins, is employed alone or optionally
in combination with another to separate/isolate the desired antibody from
the culture.

[0133] Furthermore, the technology of creating a transgenic animal may be
used to produce a transgenic animal that is a host animal having the gene
integrated into an endogenous gene, such as a transgenic bovine, a
transgenic goat, a transgenic sheep or a transgenic pig (Wright, G., et
al., (1991) Bio/Technology 9, 830-834) and a large amount of a monoclonal
antibody derived from the antibody gene can be obtained from the milk
secreted from the transgenic animal. The culture of a hybridoma in vitro
can be done by using a known nutrient medium or any nutrient medium
derivatively prepared from known basic media as used to grow, maintain
and store the hybridoma and to produce a monoclonal antibody in the
supernatant, depending on the properties of the cultured hybridoma, the
purpose of the study and the culture method.

4. Antibody Properties

(1) Agonistic Antibodies

[0134] The mutant of the agonistic antibody according to the present
invention may activate the immune system without injuring immunocompetent
cells, since it has an ADCC and/or CDC activity equal to or lower than
the original antibody, while keeping an agonistic activity. It is thus
expected that the mutant exhibits the immunoactivating action which is
equal to or higher than the original antibody and the cytotoxicity to
CD40-expressing cells which is equal to or lower than the original
antibody.

(2) Antagonistic Antibodies

[0135] The mutant of the antagonistic anti-CD40 antibody according to the
present invention has the reduced ADCC and/or CDC activity compared to
the unmodified antibody, while keeping a suppressive activity against
immunoactivating signals induced by CD40L. It is also expected to
decrease the activity of signal induction in vivo which is considered to
occur via Fc receptors.

5. Pharmaceutical Compositions

[0136] A pharmaceutical composition containing a formulation of the
purified antibody according to the present invention is also within the
scope of the present invention. The pharmaceutical composition may
preferably contain a physiologically acceptable diluent or carrier in
addition to the antibody, and may be a mixture thereof with a different
antibody or a different drug such as an antibiotic agent. The suitable
carrier may include, but not limited to, physiological saline, phosphate
buffered physiological saline, phosphate buffered physiological saline
glucose solution and buffered physiological saline. Alternatively, the
antibody may be freeze-dried for storage and when it is used,
reconstituted in an aqueous buffer as described above. The pharmaceutical
composition may be administered via the oral route, or the parenteral
route, such as intravenous, intramuscular, subcutaneous or
intraperitoneal injection or dosing.

[0137] A single effective dose, which is a combination of the antibody of
the present antibody with a suitable diluent and a physiologically
acceptable carrier, is from 0.0001 mg to 100 mg per kg of body weight,
and it may be taken at a time interval of from 2 days to 8 weeks.

[0138] When the pharmaceutical composition of the present antibody is an
agonistic antibody, it is used as: immunostimulant (antiviral or
anti-infective agent) for pathogens including, for example, hepatitis A,
B, C, D or E virus, HIV, influenza virus, simple herpes virus,
cytomegalovirus, EB virus, papiloma virus, chlamydia, mycoplasma,
toxoplasma, malaria, trypanosome and tubercle bacillus; antitumor agent
for malignant tumors having cancer cells with CD40 expressed, including,
for example, pancreatic cancer, bladder cancer, lymphoma (e.g., Hodgkin's
lymphoma), leukemia, malignant melanoma, pancreatic cancer, lung cancer,
ovarian cancer, bladder cancer, breast cancer, colon cancer, prostatic
cancer, and head and neck cancer; and therapeutic agent for autoimmune
diseases such as rheumatism. The pharmaceutical composition may be used
for a combination of the above diseases. It may be also used in
combination as adjuvant for a cancer-specific peptide. When the
pharmaceutical composition is an antagonistic antibody, on the other
hand, it is useful as: immunosuppressant in organ transplantation
(preventive or therapeutic agent for rejection in transplantation of
pancreatic islets, kidney or something else, or GVHD), therapeutic agent
for autoimmune diseases (e.g., rheumatism, psoriasis, chronic ulcerative
colitis, Crohn's disease, systemic lupus erythematosus, multiple
sclerosis, myasthenia, scleroderma, antiphospholipid antibodies syndrome,
autoimmune hepatitis, idiopathic thrombocytopenic purpura, Behcet's
syndrome, arteriosclerosis, nephritis and respiratory distress syndrome),
therapeutic agent for allergy (e.g., asthma), and therapeutic agent for
blood clotting factor VIII inhibition. The pharmaceutical composition may
be used for a combination of the above diseases.

6. Epitopes

[0139] The binding epitopes of CD40 were determined for the KM341-1-19 and
2105 antibodies having a superior agonistic activity, and for the 4D11
antibody having a superior antagonistic activity, respectively (Example
2). The present invention provides antibodies having an agonistic or
antagonistic activity which have a different variable region sequence
from those of the above antibodies but recognize the same epitope as one
of the above antibodies. These antibodies can be obtained in such a
procedure as described below.

[0140] When it is intended to acquire an agonistic anti-CD40 antibody
recognizing the same epitope as the KM341-1-19 antibody, for example,
mice or the likes are immunized with CD40 to provide monoclonal
antibodies, from which some monoclonal antibodies competing with the
KM341-1-19 antibody to bind to CD40 are screened according to the
standard procedure. From the screened antibodies, an antibody having the
same pattern of binding to the peptide as the KM341-1-19 antibody is
selected according to the method described in Example 2.

[0141] The present invention will be described in more detail below with
reference to examples. However, the present invention is not limited to
embodiments described in the examples.

Example 1

Expression and Purification of Antibody and Antigen Proteins

[0142] A vector plasmid containing a variable region of an antibody was
transfected into CHO cells (ATCC), and antibody-expressing cells were
selected by G418 to prepare a stable expression cell line.

[0143] A mutant antigen was expressed by transiently introducing a vector
into HEK cells (ATCC).

[0144] An anti-CD40 antibody was purified from the above culture
supernatant by the following method. The culture supernatant containing
an anti-CD40 antibody was affinity purified in a Hyper D® Protein A
column (manufactured by NGK Insulators, Ltd.) or in case of mouse IgG1
purification, a Protein G column (Amersham Pharmacia Biotech) according
to the attached instruction using PBS(-) as an adsorption buffer and a
0.1 M sodium citrate buffer (pH 3) as an elution buffer. The eluted
fraction was adjusted to about pH 7.2 by addition of a 1 M Tris-HCl (pH
8.0) or Na2HPO4 solution. The prepared antibody solution was substituted
with PBS(-) using a dialysis membrane (10,000 cuts, manufactured by
Spectrum Laboratories, Inc.) or an SP column (Amersham Pharmacia
Biotech), and filtered and sterilized using a membrane filter
MILLEX®-GV with a pore diameter of 0.22 μm (manufactured by
Millipore Corp.). The concentration of the purified antibody was
calculated by measurement of the absorbance at 280 nm, taking 1 mg/ml as
1.450D.

Example 2

Determination of Epitopes

[0145] A 13-mer peptide covering amino acid 175 (SEQ ID NO: 1) in an
extracellular region of CD40 was shifted by two amino acids each to
synthesize 82 peptides in total (SEQ ID NOS: 49 to 130) as spots from the
C-terminal on a cellulose membrane and acetylate the N-terminal thereof
(Jerini AG, Germany). The reaction thereafter was carried out based on a
conventional Western analysis (see Reineke, U. et al. (2001), "Epitope
mapping with synthetic peptides prepared by SPOT synthesis." Antibody
Engineering (Springer Lab Manual) Eds.: Kontermann/Dubel, 433-459, for
example). In the analysis, coloring intensity of each spot was quantified
using LumiImager® (Boehringer-Mannheim Corp.) (FIGS. 1A-1, A-2, B-1
and B-2).

[0147] In order to confirm the binding site of the anti-CD40 antibody, a
CD40-FC fusion protein having a mutation introduced thereinto was
prepared, and the binding ability thereto was examined by ELISA. Since
the anti-CD40 antibody does not cross-react with mouse B cells, five
CD40Fc fusion proteins were prepared by partially converting the amino
acid sequence into that of mouse CD40. Binding of the antibody to the
antigens was examined. The method for preparing the mutant CD40-FC fusion
proteins is shown below. The mutation site was prepared by introducing a
mouse CD40 sequence into a part to which the antibody strongly binds of
the peptide sequence. CD40mut1 converted EFTE at a site corresponding to
the 15th peptide into ALEK, CD40mut2 converted LDT at a site
corresponding to the 21st peptide into SAQ, CD40mut3 converted TH at a
site corresponding to the 24th peptide into IR, CD40mut4 converted EEGW
at a site corresponding to the 42nd peptide into KEGQ, and CD40mut5
converted VSSA at a site corresponding to the 64th peptide into QSSL. The
mutants were prepared according to a gene engineering technique (FIGS.
2A, B and C). The analysis results confirmed that the 2105 antibody has
extremely reduced binding ability to CD40mut1. The results also confirmed
that the 4D11 antibody and 2B11 have reduced binding ability to CD40mut2.

Example 3

Binding Activity of Anti-CD40 Agonistic Antibody to Ramos Cells

[0148] A Ramos cell line was suspended in a PBS staining buffer (SB)
containing 0.1% NaN3 and 2% FCS at a concentration of
2×106/ml. The cell suspension (100 μl/well) was dispensed
to a 96-well round-bottom plate (manufactured by Becton, Dickinson and
Company). Each hybridoma culture supernatant (50 μl) was added, and
incubated at an ice temperature for 30 minutes. A human IgG1 antibody to
human serum albumin as a negative control was adjusted to a concentration
of 2 μg/ml in a hybridoma culture medium, added in an amount of 50 and
then incubated at an ice temperature for 15 minutes. After washing the
plate with SB, 50 μl of a 250-fold diluted R-PE fluorescently labeled
anti-human antibody (manufactured by Southern Biotechnology Associates,
Inc.) was added, and incubated at an ice temperature for 15 minutes.
After washing the plate with SB twice, it was suspended in 300 to 500
μl of a FACS buffer, and fluorescence intensity of each cell was
measured using FACS (FACSort, FACScan, manufactured by Becton, Dickinson
and Company).

[0149] 5.0×105 cells/ml of a Ramos cell suspension was seeded
on a 96-well plate at 100 μl/well. A hybridoma culture supernatant or
purified antibody was diluted to 20 μg/ml in a medium, and the
dilution was added to the 96-well plate at a concentration of 100
μl/well. After culturing overnight, cells were harvested, and R-PE
labeled anti-CD95 antibody (Pharmingen NJ) was used for the cells.
Analysis was carried out using FACScan or FACSsort (Becton, Dickinson and
Company).

[0150] 1.0×106 cells/ml of a Ramos cell suspension was seeded
on a 96-well plate at 50 μl/well. A hybridoma culture supernatant or
purified antibody was adjusted to 2 μg/ml in a medium, and the medium
was added to the 96-well plate at 100 μl/well. 4 μg/ml of a soluble
CD40 ligand (Alexis Corporation) and 4 μg/ml of an anti-FLAG antibody
(M2, Sigma) were added to a medium, and the medium was added to a 96-well
plate at 50 μl/well. After culturing overnight, cells were harvested,
and an R-PE labeled anti-CD95 antibody (Pharmingen NJ) was used for the
cells. Analysis was carried out using FACS.

Example 6

Measurement of CDC Activity in Anti-CD40 Antibody

[0151] In the CDC assay, 2,000 Cr51-labeled target cells and a human
serum-derived complement (manufactured by Sigma Co.) or rabbit
serum-derived complement (Cedarlane Laboratories Limited, Ontario,
Canada) at a final concentration of 5% were cultured in a round-bottom
96-well plate in a total volume of 200 μL together with the antibody
at various concentrations at 37° C. in the presence of 5% CO2
for two hours.

[0152] After culturing, the plate was centrifuged to cause the cells to
precipitate, and then 50 μL of the supernatant was transferred to a
96-well plate including a powder scintillator (Lumaplate®-96:
manufactured by Packard Instrument Co., Inc.) and dried at 55° C.
for 1.5 hours. After confirming that the plate was dried, it was covered
with a special cover (TopSeal®-A: 96-well microplates: manufactured by
Packard Instrument Co., Inc.), and the γ-ray dose was measured with
a scintillation counter (TopCount: manufactured by Packard Instrument
Co., Inc.).

Example 7

Measurement of ADCC Activity of Anti-CD40 Antibody

[0153] As antibody-mediated cytotoxicity, cytotoxicity to target cells in
the presence of cells having killer activity such as NK cells or
neutrophils and an antibody (Antibody-Dependent Cellular Cytotoxicity,
hereinafter ADCC), and cytotoxicity to target cells in the presence of a
complement and an antibody (Complement-Dependent Cytotoxicity,
hereinafter CDC) were measured. hIgG was used as a control.

[0154] The measurement method is simply described as follows. Radioactive
chromium (Cr51) was incorporated into the cytoplasm of the target
cells, and the amount of Cr51 released in the culture solution by
cell death was measured as a γ-ray dose.

[0155] Specifically, 105 target cells of a Burkitt's lymphoma cell
line Raji (ATCC CCL-86) were suspended in 15 μL of fetal calf serum
(FCS). 50 μL (37 MBq/mL) of Cr51-labeled sodium chromate
(manufactured by PerkinElmer, Inc.: hereinafter referred to as Cr51)
was added to the suspension, and the cells were cultured at 37° C.
for one hour. Next, 10 mL of a medium was added, and the medium was
discarded by centrifugation. This operation was repeated three times to
remove Cr51 not incorporated in the cells.

[0156] In the ADCC assay, 2,000 Cr51-labeled target cells and 200,000
healthy human peripheral blood mononuclear leukocytes obtained by the
method described in Example 6 were cultured in a round-bottom 96-well
plate (manufactured by Falcon) in a total volume of 200 μL together
with the antibody at various concentrations at 37° C. in the
presence of 5% CO2 for four hours.

[0157] After culturing, the plate was centrifuged to cause the cells to
precipitate, and then 50 μL of the supernatant was transferred to a
96-well plate including a powder scintillator (Lumaplate®-96:
manufactured by Packard Instrument Co., Inc.) and dried at 55° C.
for 1.5 hours. After confirming that the plate was dried, the plate was
covered with a special cover (TopSeal®-A: 96-well microplates:
manufactured by Packard Instrument Co., Inc.), and the γ-ray dose
was measured with a scintillation counter (TopCount: manufactured by
Packard Instrument Co., Inc.).

[0158] Gene cloning of anti-CD40 agonistic antibodies KM341-1-19 and 2105
is described in WO 02/099186. It is reported that CDC activity is reduced
by converting Pro at position 331 in the IgG2 constant region into Ser.
To reduce CDC activity of the KM341-1-19 antibody and the 2105 antibody,
a P331S mutation was introduced into the IgG2 constant region thereof.

[0159] The human IgG1 constant region of an antibody-expressing vector
N5KG1-Val Lark (IDEC Pharmaceuticals: hereinafter abbreviated to N5KG1)
was substituted with human IgG2 to prepare N5KG2, and the Pro at position
331 of IgG2 was converted into Ser to prepare a mutation. cDNA cloning of
the IgG2 constant region was carried out by harvesting KM341-1-19
hybridoma by centrifugation, adding TRIZOL (Gibco BRL), and extracting
total RNA according to the instruction. The antibody cDNA variable region
was cloned using a SMART RACE cDNA amplification kit of Clontech
Laboratories, Inc. according to the attached instruction. 1st strand cDNA
was prepared using 5 μg of total RNA as a template. PCR was carried
out with tnIgG3Nhe: atatGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGC (SEQ ID NO:
2)G and tnIgG2Bam: atatggatccTCATTTACCCGGAGACAGGGAGAGGCTC (SEQ ID: 3) as
primer sequences using a ZtaqPCR kit (Takara) in 30 cycles each
consisting of reaction at 98° C. for 1 second, at 55° C.
for 30 seconds and at 72° C. for 1 minute to amplify the gene.
After the reaction, the amplified product was purified by a QIAGEN PCR
purification kit, digested with NheI and BamHI, and incorporated into
N5KG1 to confirm the sequence. This vector was defined as N5KG2.

[0160] N5KG2Ser (with Pro at position 331 converted into Ser) was prepared
as follows. Reaction at 98° C. for 1 second, at 60° C. for
30 seconds and at 72° C. for 30 seconds was carried out 15 times
using N5KG2 as a template and primers IgG3Nhe:
atatGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCG (SEQ ID NO: 4) and G2Ser2:
GTTTTCTCGATGGAGGCTGGGAGGCC (SEQ ID NO: 5). At the same time, reaction at
98° C. for 1 second, at 60° C. for 30 seconds and at
72° C. for 30 seconds was carried out 15 times using N5KG2 as a
template and primers IgG2Bam: atatggatccTCATTTACCCGGAGACAGGGAGAGGCTC (SEQ
ID NO: 6) and G2Ser1: GGCCTCCCAGCCTCCATCGAGAAAAC (SEQ ID NO: 7). The
amplified DNA fragments were purified using a PCR purification kit, and
the same amounts of the two purified DNA fragments were mixed.
Thereafter, reaction at 98° C. for 1 second, at 60° C. for
30 seconds and at 72° C. for 30 seconds was carried out five
times. Primers IgG3Nhe and IgG2Bam were added to the mixture, and the
same reaction was carried out 15 times. The amplified DNA fragment was
cleaved with NheI and BamHI, and substituted with the IgG1 constant
region of the N5KG1 vector (N5KG2Ser). The fragment containing the
sequence of the antibody variable region digested with BglII and NheI was
incorporated into the N5KG2Ser vector.

[0161] The antibody expressed and purified by the above method was
evaluated in terms of binding ability to Ramos cells (FIG. 3A) and
agonistic activity (FIG. 3B). The fluctuation in activity due to
introduction of the P331S variation was not observed.

[0162] CDC activity was measured by the above method. A rabbit
serum-derived complement was used, and Ramos cells were used as target
cells. The results confirmed that, in a KM341-1-19 antibody at an
antibody concentration of 1 μg/ml, IgG2ser exhibited CDC activity
significantly reduced as compared with IgG2 (FIG. 4A). On the other hand,
when a human supplement was used, no change was observed (FIG. 4B).

[0163] Among the anti-CD antibodies described in WO 02/088186, two
antibodies exhibiting strongest agonistic activity (KM341-1-19 antibody
and 2105 antibody) belong to IgG2 subclass. In order to examine whether
or not the IgG2 subclass is important for activation of CD40, recombinant
proteins having an antibody constant region converted into IgG1, IgG3 and
IgG4, respectively, were prepared, and measured in terms of binding
ability to an antigen and CD95 expression enhancing activity in Ramos
cells according to Examples 4 and 6. IgG1 was expressed using N5KG1, and
IgG2 and IgG3 were respectively expressed using expression vectors N5KG2
and N5KG3 obtained by substituting the N5KG1 constant region with IgG2
and IgG3, respectively. cDNA cloning of the IgG3 constant region was
carried out according to the IgG2 cloning method partially modified,
using an IgG3-specific primer. IgG4 was expressed using N5KG4PE (IDEC
Pharmaceuticals).

[0164] The antibody protein was expressed according to Example 1. Binding
activity to Ramos cells expressing human CD40 of the KM341-1-19 antibody
and the 2105 antibody was not affected by converting IgG2 into IgG1, IgG3
or IgG4 (FIGS. 5A-1 and 5A-2). However, these antibodies were found to
have CD95 expression enhancing activity in Ramos cells reduced by 10% or
more (FIGS. 5B-1 and 5B-2). This shows that not only the structure of the
variable region defining the binding region of the antibody but also the
structure of the constant region of the antibody are important for the
strong agonistic activity of the 2105 antibody and the KM341-1-19
antibody. Thus, in order to examine which region in the IgG2 constant
region is important for agonistic activity, a domain swap mutant in which
the IgG2 structure is mixed with the IgG4 structure was prepared to
measure its activity. As described below, a domain swap mutant is
prepared by substitution of a hinge region. In this case, the "hinge
region" includes the upper hinge (from Kabat EU code 216), middle hinge
(from Kabat EU code 226) and lower hinge (Kabat EU code 231), as
described Ole H Brekke et. al., Immunology Today 1995, 16, 85-90. Four
domain swap mutants IgG2/4 (CH1 and hinge region: IgG2, other regions:
IgG4), IgG4/2/4 (hinge region: IgG2, other regions: IgG4), IgG2/4/4 (CH1:
IgG2, other regions: IgG4) and IgG4/2/2 (CH1: IgG4, other regions: IgG2)
were prepared respectively for the KM341-1-19 antibody and the 2105
antibody.

[0165] A vector N5KG2/4 for expressing the IgG2/4 antibody was prepared
using a Ztaq PCR kit (Takara). Reaction at 98° C. for 1 second, at
60° C. for 30 seconds and at 72° C. for 30 seconds was
carried out 15 times using N5KG2 as a template and primers IgG3Bam:
atatggatccTCATTTACCCGGAGACAGGGAGAGGC (SEQ ID NO: 8) and 24Chi4:
AGGGGTCCGGGAGATCATGAGAGTGTCCTT (SEQ ID NO: 9). At the same time, reaction
at 98° C. for 1 second, at 60° C. for 30 seconds and at
72° C. for 30 seconds was carried out 15 times using N5KG4 (IDEC
Pharmaceuticals) as a template and primers 24Chi3:
AAGGACACTCTCATGATCTCCCGGACCCCT (SEQ ID NO: 10) and linkH2:
tgatcatacgtagatatcacggc (SEQ ID NO: 11). The amplified DNA fragments were
purified using a PCR purification kit, and the same amounts of the two
purified DNA fragments were mixed. Thereafter, reaction at 98° C.
for 1 second, at 60° C. for 30 seconds and at 72° C. for 30
seconds was carried out five times. Primers IgG3Bam and linkH2:
tgatcatacgtagatatcacggc (SEQ ID NO: 12) were added to the mixture, and
the same reaction was carried out 15 times. The amplified DNA fragment
was cleaved with NheI and BamHI, and substituted with the IgG1 constant
region of the N5KG1 vector.

[0166] A vector N5KG4/2/4 for expressing IgG4/2/4 was prepared as follows.
Reaction at 98° C. for 1 second, at 60° C. for 30 seconds
and at 72° C. for 30 seconds was carried out 15 times using N5KG4
as a template and primers linkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO:
13), G2Hin3: TTTGCGCTCAACTGTCTTGTCCACCTTGGTGTTGCTGGG (SEQ ID NO: 14),
linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 15) and G2Hin4:
ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCG (SEQ ID NO: 16). The amplified DNA
fragments were purified with a PCR purification kit, and the same amounts
of the two purified DNA fragments were mixed. Thereafter, reaction at
98° C. for 1 second, at 60° C. for 30 seconds and at
72° C. for 30 seconds was carried out five times using the mixture
as a template. Primers linkH and linkH2 were added to the mixture, and
the same reaction was carried out 15 times. The amplified DNA fragment
was cleaved with NheI and BamHI, and substituted with the IgG1 constant
region of the N5KG1 vector.

[0167] A vector N5KG2/4/4 for expressing IgG2/4/4 was prepared as follows.
Reaction at 98° C. for 1 second, at 60° C. for 30 seconds
and at 72° C. for 30 seconds was carried out 15 times using N5KG2
as a template and primers linkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO:
17) and G4CH1-2: GGTGTTGCTGGGCTTGTGATCTACGTTGCAG (SEQ ID NO: 18). At the
same time, reaction at 98° C. for 1 second, at 60° C. for
30 seconds and at 72° C. for 30 seconds was carried out 15 times
using N5KG4 as a template and primers G4CH1-1:
CTGCAACGTAGATCACAAGCCCAGCAACACC (SEQ ID NO: 19) and linkH2:
tgatcatacgtagatatcacggc (SEQ ID NO: 20). The amplified DNA fragments were
purified using a PCR purification kit, and the same amounts of the two
purified DNA fragments were mixed. Thereafter, reaction at 98° C.
for 1 second, at 60° C. for 30 seconds and at 72° C. for 30
seconds was carried out five times. Primers linkH and linkH2 were added
to the mixture, and the same reaction was carried out 15 times. The
amplified DNA fragment was cleaved with NheI and BamHI, and substituted
with the IgG1 constant region of the N5KG1 vector.

[0168] A vector N5KG4/2/2 for expressing IgG4/2/2 was prepared as follows.
Reaction at 98° C. for 1 second, at 60° C. for 30 seconds
and at 72° C. for 30 seconds was carried out using N5KG4 as a
template and primers linkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 21)
and G4CH1-2: GGTGTTGCTGGGCTTGTGATCTACGTTGCAG (SEQ ID NO: 22). At the same
time, reaction at 98° C. for 1 second, at 60° C. for 30
seconds and at 72° C. for 30 seconds was carried out 15 times
using N5KG2 as a template and primers G4CH1-1:
CTGCAACGTAGATCACAAGCCCAGCAACACC (SEQ ID NO: 23) and linkH2:
tgatcatacgtagatatcacggc (SEQ ID NO: 24). The amplified DNA fragments were
purified using a PCR purification kit, and the same amounts of the two
purified DNA fragments were mixed. Thereafter, reaction at 98° C.
for 1 second, at 60° C. for 30 seconds and at 72° C. for 30
seconds was carried out five times. Primers linkH and linkH2 were added
to the mixture, and the same reaction was carried out 15 times. The
amplified DNA fragment was cleaved with NheI and BamHI, and substituted
with the IgG1 constant region of the N5KG1 vector.

[0169] Binding activity of the respective four domain swap mutants of the
KM341-1-19 antibody and the 2105 antibody was examined. As a result, no
difference between them and the original IgG2 was observed in terms of
binding ability (FIGS. 6A-1 and 6A-2). However, only IgG2/4/4 of both the
KM341-1-19 antibody and the 2105 antibody exhibited significantly reduced
agonistic activity (FIGS. 6B-1 and 6B-2). The results confirmed that the
hinge region of IgG2 is important for agonistic activity.

[0171] N5KG2UH4 was prepared as follows. Reaction at 98° C. for 1
second, at 60° C. for 30 seconds and at 72° C. for 30
seconds was carried out 15 times using N5KG2 as a template and primers
linkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 25) and UH4-2:
CACAACATTTggaCTCAACTcCTTGTCCACC (SEQ ID NO: 26). At the same time,
reaction at 98° C. for 1 second, at 60° C. for 30 seconds
and at 72° C. for 30 seconds was carried out 15 times using N5KG2
as a template and primers UH4-1: GGTGGACAAGAgAGTTGAGtccAAATGTTGTG (SEQ ID
NO: 27) and linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 28). The
amplified DNA fragments were purified using a PCR purification kit, and
the same amounts of the two purified DNA fragments were mixed.
Thereafter, reaction at 98° C. for 1 second, at 60° C. for
30 seconds and at 72° C. for 30 seconds was carried out five
times. Primers linkH and linkH2 were added to the mixture, and the same
reaction was carried out 15 times. The amplified DNA fragment was cleaved
with NheI and BamHI, and substituted with the IgG1 constant region of the
N5KG1 vector.

[0172] N5KG2 MH4 was prepared as follows. Reaction at 98° C. for 1
second, at 60° C. for 30 seconds and at 72° C. for 30
seconds was carried out 15 times using N5KG2 as a template and primers
linkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 29) and UM4-2:
GGCACGGTGGGCAtgggggaccataTTTGCGCTC (SEQ ID NO: 30). At the same time,
reaction at 98° C. for 1 second, at 60° C. for 30 seconds
and at 72° C. for 30 seconds was carried out 15 times using N5KG2
as a template and primers UM4-1: GAGCGCAAAtatggtcccccaTGCCCACCGTGCC (SEQ
ID NO: 31) and linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 32). The
amplified DNA fragments were purified using a PCR purification kit, and
the same amounts of the two purified DNA fragments were mixed.
Thereafter, reaction at 98° C. for 1 second, at 60° C. for
30 seconds and at 72° C. for 30 seconds was carried out five
times. Primers linkH and linkH2 were added to the mixture, and the same
reaction was carried out 15 times. The amplified DNA fragment was cleaved
with NheI and BamHI, and substituted with the IgG1 constant region of the
N5KG1 vector.

[0173] N5KG2LH4 was prepared as follows. Reaction at 98° C. for 4
second, at 60° C. for 30 seconds and at 72° C. for 30
seconds was carried out 15 times using N5KG2 as a template and primers
linkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 33) and UL4-2:
GAAGACTGACGGTCCccccaggaactcTGGTGCTGGGCA (SEQ ID NO: 34). At the same
time, reaction at 98° C. for 1 second, at 60° C. for 30
seconds and at 72° C. for 30 seconds was carried out 15 times
using N5KG2 as a template and primers UL4-1:
TGCCCAGCACCAgagttcctggggGGACCGTCAGTCTTC (SEQ ID NO: 35) and linkH2:
tgatcatacgtagatatcacggc (SEQ ID NO: 36). The amplified DNA fragments were
purified using a PCR purification kit, and the same amounts of the two
purified DNA fragments were mixed. Thereafter, reaction at 98° C.
for 1 second, at 60° C. for 30 seconds and at 72° C. for 30
seconds was carried out five times. Primers linkH and linkH2 were added
to the mixture, and the same reaction was carried out 15 times. The
amplified DNA fragment was cleaved with NheI and BamHI, and substituted
with the IgG1 constant region of the N5KG1 vector.

[0174] The three respective domain swap mutants of the KM341-1-19 antibody
and the 2105 antibody were examined to have the same binding activity to
an antigen (FIGS. 6A-1 and 6A-2). However, IgG2UH4 and IgG2 MH4 exhibited
significantly reduced agonistic activity to Ramos cells (FIGS. 6B-1 and
6B-2). It was found from the above that the structures of upper hinge and
middle hinge in the hinge region are important for IgG2
subclass-dependent agonistic activity of the anti-CD40 antibodies
KM341-1-19 and 2105.

[0175] Since IgG2 subclass was found to be important for agonistic
activity, antibodies of subclass other than IgG2 were converted to those
of IgG2 subclass to examine whether or not the agonistic activity was
enhanced. In the examination on several clones, agonistic activity of F76
could be enhanced by converting IgG1 subclass to IgG2 subclass (FIGS. 7A
and B).

Example 11

Preparation of Anti-CD40 Antagonist Antibody Mutants

[0176] A DNA fragment containing a heavy chain and a light chain of a 4D11
antibody gene described in WO 02/088186, whose original subclass is IgG1,
was digested with BglII and NheI, purified, and then integrated into
N5KG4PE, N5KG4P and N5KG4 vectors (IDEC Pharmaceuticals). N5KG4PE
contains point mutations S228P and L235E in the IgG4 constant region, and
N5KG4P contains a point mutation S228P in the IgG4 constant region. The
antibody protein was expressed and purified according to the above
method. The antibody was purified according to the above method using
binding to Ramos cells as an index. Change in binding activity of IgG1,
IgG4, IgG4P and IgG4PE to Ramos cells was not observed (FIG. 8A).
Antagonistic activity of IgG1 was compared with those of various IgG4
mutants according to the above method to find that antagonistic activity
of IgG1 does not differ from those of the IgG4 mutants (FIG. 8B).

[0177] ADCC activity and CDC activity of anti-CD40 mutant antibodies were
evaluated according to the above method.

[0178] When using human MNC as effector cells and CD40-expressing Daudi
cells as target cells, two mutants IgG4 and IgG4PE were respectively
observed to have ADCC activity significantly reduced as compared with
IgG1 as the original subclass of the 4D11 antibody (FIG. 9).

[0179] CDC activity of IgG1 was compared with that of IgG4P using Daudi
cells as target cells. IgG4P was found to have CDC activity significantly
reduced as compared with IgG1 (FIG. 10).

Example 13

Effect of Anti-CD40 Antagonistic Antibody on B Cells

[0180] 100 μg each of IgG1, IgG4P and IgG4PE of the 4D11 antibody was
administered to the tail vein of mice having a genetic background whereby
they were homozygotes for mouse endogenous disrupted CD40 and harboring a
transgene of a human CD40 gene (Yasui. et al. Int. Immunol. 2002 Vol 14:
319). 24 hours after the administration, blood was collected from the
orbital venous plexus. After hemolysis with 0.16 mol/L of ammonium
chloride, an FITC-labeled anti-B220 antibody was added to the hemolysate,
and it was analyzed using FACS. The results are shown in FIG. 11. In the
figure, the longitudinal axis indicates the ratio of B cells in the total
lymphocytes. IgG1 reduced the ratio of B cells most, IgG4P reduced the
ratio to a lesser extent, and IgG4PE reduced the ratio to a much lesser
extent. 24 hours after the administration, the spleen was removed and
crushed with a slide glass to prepare a cell suspension. After hemolysis
of the cell suspension, a PE-labeled anti-B220 antibody and an
FITC-labeled anti-CD23, CD86 or CD95 antibody were used for the
hemolysate, and it was analyzed using FACS. The results are shown in
FIGS. 12A, B and C. In the figures, the longitudinal axis indicates the
ratio of B cells expressing each surface marker in the total lymphocytes.
4D11G1 was found to achieve the same level of increase in expression of
each marker as in a commercially available mouse anti-human CD40
agonistic antibody 5C3 (Pharmingen). IgG4PE achieved a smaller increase
in expression of each activation surface marker as compared with IgG1 and
IgG4P.

Example 14

Effect of Inhibiting Production of Antigen-Specific Antibody and Change in
the Number of B Cells Caused by Anti-CD40 Antagonistic Antibody

[0181] 100 μg (based on NP-CGG) of a complex of
4-hydroxy-3-nitrophenylacetyl-chiken γ-globulin conjugates (NP-CGG:
distributed by Professor Hitoshi KIKUTANI, Research Institute for
Microbial Diseases, Osaka University) and alum (aluminum hydroxide gel)
was intraperitoneally administered to mice having a genetic background
whereby they were homozygotes for mouse endogenous disrupted CD40 and
harboring a transgene of a human CD40 gene (Yasui et al. Int. Immunol.
2002 Vol 14: 319) to sensitize the mice. Immediately before the antigen
sensitization, 50 or 100 μg of each antibody was administered to the
tail vein. 100 μg of an anti-human albumin human IgG4PE antibody was
administered as a negative control. 7 and 14 days after the
sensitization, blood was collected from the orbital venous plexus. The
amounts of NP-specific IgG1 and IgM antibodies in the serum were measured
by the ELISA method. The ELISA method was carried out as follows. 50
μl/well of NP-bound bovine serum albumin (NP-BSA: 2.5 μg/ml) was
added to each well of a 96-well microplate for ELISA (Maxisorp,
manufactured by Nunc A/S) and incubated at 4° C. to cause NP-BSA
to be adsorbed thereon. Next, the supernatant was discarded, and a
blocking reagent (SuperBlock, manufactured by Pierce Biotechnology, Inc.)
was added to each well and incubated at room temperature to carry out
blocking. Then, each well was washed with a phosphate buffer (PBS-T)
containing 0.1% Tween 20 three times. Next, each serum diluted with PBS-T
containing 10% Block Ace (50 μl/well) was added to each well, and
incubated and reacted at 37° C. for two hours. The microplate was
washed with PBS-T three times. Then, a 1,000-fold dilution of a goat
anti-mouse IgG1 antibody or IgM antibody labeled with alkaline
phosphatase (Cosmo Bio, 1070-04 or 1020-04) with PBS-T containing 10%
Block Ace (50 μg/well) was added to each well, and incubated at
37° C. for two hours. Next, the microplate was washed with PBS-T,
and then a coloring substrate solution (50 μl/well, Sigma 104,
phosphatase substrate) was added to each well. The absorbance at a
wavelength of 405 nm was measured using a microplate reader. The results
are shown in FIGS. 13A and B. In the figures, the longitudinal axis
indicates values obtained by converting a 10,000-fold dilution (in the
case of IgG1) or 100-fold dilution (in the case of the IgM antibody) of
serum collected from C57BL/6 mice, to which NP-CGG was injected twice,
and pooled into one unit. The 4D11 antibody and the IgG4P or IgG4PE
antibody of 281 inhibited production of NP-specific IgG1 and IgM
antibodies equally strongly.

[0182] The change in the number of B cells in the peripheral blood and
spleen in the mice used for examining the effect of inhibiting antibody
production was measured according to the same method as in Example 1. The
results are shown in FIGS. 14A and B. The 4D11 antibody and the IgG4P
antibody of 281 reduced the ratio of B cells in peripheral blood
significantly as compared with the IgG4PE antibody. Administration of 100
μg of the IgG4PE antibody did not change the ratio of B cells in the
spleen removed 14 days after the antigen sensitization. However,
administration of IgG4P changed or tended to change the ratio.

Example 15

Effect of Anti-CD40 Antagonistic Antibody on Cynomolgus Monkeys

[0183] 30 mg/kg of IgG4P or IgG4PE of a 4D11 antibody was administered to
the forearm cephalic vein of cynomolgus monkeys, and blood was collected
from the femoral vein after a certain period of time. In the subset
analysis of peripheral blood lymphocytes, an FITC-labeled anti-CD3
antibody, PE-labeled anti-CD20 antibody and APC-labeled anti-CD45
antibody were used for each cell suspension, and the ratio of positive
cells was measured using FACS to calculate the ratio of CD45 positive
cells. The results are shown in FIG. 15. In the figure, the longitudinal
axis indicates the ratio of CD20 positive cells at each time to CD20
positive cells before antibody administration. 1 to 7 days after the
antibody administration, CD20 positive cells were reduced by about 40% in
individuals to which the IgG4P antibody was administered. However, 4 days
after the administration, CD20 positive cells were reduced by only about
20% in individuals to which the IgG4PE antibody was administered.

[0184] The IL12 concentration in serum was measured by the ELISA method.
Blood collected from the femoral vein was allowed to stand at room
temperature for 20 to 60 minutes, and then centrifuged at 3,000 rpm at
room temperature for 15 minutes. The IL12 concentration in the resulting
serum was measured using a monkey IL12 ELISA kit (BioSource International
Inc.). The results are shown in FIG. 16. No increase in IL12 production
by the IgG4PE antibody was observed at any blood collection point.
However, maximum IL12 production by the IgG4P antibody was observed on
the 4th day.

[0185] Nine male cynomolgus monkeys were intradermally and intramuscularly
sensitized with Tetanus toxoid (TTx) (10 Lf/ml; Denka Seiken Co., Ltd.)
to induce delayed hypersensitivity to TTx. At the same time, 10 minutes
before the start of sensitization, 0.1 and 10 mg/kg of a 4D11G4PE
antibody was intravenously administered to each three animals three times
(once a week) to examine the effect of 4D11G4PE on delayed
hypersensitivity. Under anesthesia by intramuscular administration of
ketamine, sensitization was carried out by intradermal administration of
TTx to the back (50×12 sites) and intramuscular administration of
TTx to the femur (0.6 mL/body), and challenge was carried out by
intradermal administration of TTx to the thorax (10 μL/site, 0 to 10
Lf/ml for each three sites) 21 days after the sensitization. 24 and 48
hours after the elicitation, skin reaction at the administration sites
was observed and evaluated according to the Draize skin irritation score.
The results of determining the TTx concentration in each three sites were
respectively a mean value. The results are shown in FIG. 17.
Administration of the 4D11G4PE antibody apparently inhibited the delayed
hypersensitivity reaction observed 24 and 48 hours after the
administration.

[0186] The effect of TTx on TTx-specific IgG and IgM antibody titers was
examined. Blood collected from the femoral vein over time was allowed to
stand at room temperature for 20 to 60 minutes, and then centrifuged at
3,000 rpm at room temperature for 15 minutes. The antibody titer in the
resulting serum was measured using the ELISA method. The ELISA method was
carried out as follows. 100 μl/well of TTx (0.5 Lf/ml) was added to
each well of a 96-well microplate for ELISA (Maxisorp, manufactured by
Nunc A/S) and incubated at 4° C. to cause TTx to be adsorbed
thereon. Next, the supernatant was discarded, and a blocking reagent
(phosphate buffer containing 0.5% BSA) was added to each well and
incubated at room temperature to carry out blocking. Then, each well was
washed with a phosphate buffer (PBS-T) containing 0.05% Tween 20 three
times. Next, each serum diluted with PBS-T containing 0.5% BSA (100 to
819,200-fold dilution, dilution magnification: 2; 100 μl/well) was
added to each well, and incubated and reacted at room temperature for two
hours. The microplate was washed with PBS-T three times. Then, a
3,000-fold dilution of a goat anti-monkey IgG antibody or IgM antibody
labeled with peroxidase (Nordic Immunology) with PBS-T containing 0.5%
BSA (100 μg/well) was added to each well, and incubated at room
temperature for one hour. Next, the microplate was washed with PBS-T, and
then a coloring substrate solution (100 μl/well, o-phenylenediamine
hydrochloride+aqueous hydrogen peroxide) was added to each well. The
absorbance at a wavelength of 492 nm was measured using a microplate
reader. The anti-TTx antibody titer was defined as a maximum dilution
magnification to make the absorbance 0.1 and more. The antibody titer was
0 when the absorbance did not reach 0.1 even at 100-fold dilution. The
results are shown in FIGS. 18 and 19. Administration of 1 mg/kg of
4D11G4PE suppressed the TTx-specific IgG and IgM antibody titers to about
1/10. When 10 mg/kg of 4D11G4PE was administered, the antibody titers
were below the detection sensitivity at any blood collection point.

[0187] Blood collected from a healthy human was divided into four aliquots
(each 6 ml). Control human IgG4PE, control mouse IgG2a, human anti-human
CD40 IgG4PE (4D11) and mouse anti-human CD154 IgG2a (5C8) were
respectively added to the fractions so that each fraction had a blood
concentration of 100 μg/ml. A flat perfusion chamber (GlycoTech Corp.)
and a collagen-coated Petri dish were assembled according to the attached
instruction. The blood treated with various antibodies was caused to flow
into the chamber at a rate that can apply a shear stress of 1,500/s to
the blood for seven minutes. Thereafter, a 4% paraformaldehyde phosphate
buffer was caused to flow into the chamber at a rate that can apply a
shear stress of 1,500/s to the buffer for 10 minutes. The platelet
aggregate formed on the Petri dish was fixed, stained with a
platelet-specific PE-labeled CD41a antibody, and observed with a
fluorescence microscope. The results are shown in FIGS. 20A and B. The
blood treated with human anti-human CD40 IgG4PE (4D11) formed a platelet
aggregate on the collagen-coated Petri dish, as the blood treated with
the control antibodies did. However, the blood treated with mouse
anti-human CD154 IgG2a did not form a platelet aggregate.

Example 18

Evaluation of Stability of Anti-CD40 Antagonistic Antibody

[0188] Constant region-modified antibodies of the 4D11 antibody were
compared and examined in terms of stability. In the evaluation method,
culture supernatants obtained by respectively transiently expressing G4P,
G4PE, G2Ser and G4/2/4 in HEK293 cells were charged with a Protein A
column (Amersham Pharmacia Biotech), eluted with a 0.1 M citrate buffer
(pH 2.7), and then incubated at 37° C. for 1 minute and 10
minutes. Thereafter, they are neutralized with a 50 nM phosphate buffer
(pH 7.0). The oligomer content in the resulting antibody solutions was
measured using a gel filtration column (Tosoh Corp.). As a result, it was
found that the oligomer content increases in proportion with the
incubation time, and G4/2/4 produces an oligomer easiest, G4PE second
easiest, G2Ser third easiest, and G4P fourth easiest (FIG. 21).

[0189] A graft collected from the tail of DBA/2 mice was grafted into the
side dorsal thorax of C57BL/6 background mice having a genetic background
whereby they were homozygotes for mouse endogenous disrupted CD40 and
harboring a transgene of a human CD40 gene, and the graft was fixed with
a plaster for seven days. 100 μg of a test substance 4D11G4PE or a
vehicle was administered to the tail vein 0, 2, 4, 7, 9, 11 and 14 days
after the skin graft, respectively. To inhibit graft rejection by NK
cells, 100 μg of an anti-asialo GML antibody was intraperitoneally
administered to all mice 3 days before the operation and 1, 4 and 9 days
after the operation. The results are shown in FIG. 22. The delay in graft
rejection was observed to be significant in the 4D11G4PE-administered
group as compared with the vehicle-administered group.

[0191] T24, Hs 766T and Capan-2 were digested with trypsin and harvested,
and Ramos was harvested as is. The cell lines were washed with PBS, and
then re-suspended in a staining buffer containing 1 μg/ml of 341G2Ser.
The staining buffer was prepared by adding 0.05 mM EDTA, 0.05% sodium
azide and 5% immobilized bovine serum to PBS. After incubation at
4° C. for 15 minutes, the cells were washed with the staining
buffer twice, and re-suspended in a 1:250 dilution of PE-bound goat
anti-human IgG (γ) (Southern Biotechnology Associates, Inc) with
the staining buffer. After incubation at 4° C. for 15 minutes, the
cells were washed with the staining buffer twice, and analyzed with
FACSCalibur® (manufactured by BD Biosciences). The same amount of a
human anti-2,4-dinitrophenol (DNP) antibody was used as a negative
control. The analysis was carried out using Cellquest® (manufactured
by BD Biosciences) as data analysis software to calculate the mean
fluorescence intensity.

[0192] As a result, Ramos, T24, Hs766T and Capan-2 had a mean fluorescence
intensity obviously higher than that of the negative control when stained
with 341G2Ser, and thus expression of CD40 was confirmed.

Example 21

Effect of Anti-CD40 Agonistic Antibody on Human Tumor Cell Lines

[0193] 2.5×103 Ramos cells, 2.5×102 T24 cells,
5×103 Hs766T cells and 5×103 Capan-2 cells were
respectively suspended in a medium to make the total volume of 100 μL
in a flat bottom 96-well plate (manufactured by Falcon). Ramos and
Hs766T, Capan-2 and T24 were cultured for 66 hours, 90 hours and 114
hours, respectively, together with 341G2Ser at a concentration of 1 ng/ml
to 1,000 ng/ml at 37° C. in the presence of 5% CO2. 10 μL
(3.7 MBq/mL) of 3H-labeled thymidine (manufactured by Amersham
Biosciences) was added and cultured at 37° C. in the presence of
5% CO2 for six hours. Ramos cells were harvested on Printed
Filtermat A (manufactured by PerkinElmer, Inc.) using a 96 micro cell
harvester (manufactured by Skatron Instruments, Inc.), and covered with a
sample bag (manufactured by PerkinElmer, Inc.). 12 mL of Betaplate Scint
(manufactured by PerkinElmer, Inc.) was added, and the β-ray dose
was measured with a liquid scintillation counter (Pharmacia 1205
Betaplate: manufactured by Pharmacia Corp.). Hs 766T cells, T24 cells and
Capan-2 cells were respectively harvested on Unifilter (manufactured by
PerkinElmer, Inc.) using a harvester (manufactured by PerkinElmer, Inc.).
A special seal was attached to the back of each filter, and 20 μL/well
of MicroScint 20 (manufactured by PerkinElmer, Inc.) was added thereto.
The β-ray dose was measured with a scintillation counter (TopCount:
manufactured by Packard Instrument Co., Inc.). Data were expressed as
cell survival rates (%) obtained by dividing a mean of triplicate
measurements obtained in three independent tests by a value of
non-treatment control.

[0194] As a result, the cell survival rates were reduced in all cell lines
depending on the 341G2Ser concentration (Table 1). When adding 100 ng/ml
of 341G2Ser, the Ramos cell survival rate was 58%, the T24 cell survival
rate was 22%, the Hs 766T cell survival rate was 15%, and the Capan-2
cell survival rate was 77%. 341G2Ser was found to have activity of
inhibiting growth of Ramos cells, T24 cells, Hs 766T cells and Capan-2
cells.

[0195] Six-week-old female Balb/c nude mice (purchased from CLEA Japan,
Inc.) were irradiated with 3Gy radiation, and 2×107
cells/mouse of Ramos cells were subcutaneously grafted into the back
thereof. 16 days after the graft, the size of tumors that took there was
measured. Cancer-bearing mice having a tumor size of 50 to 170 mm3
were classified into groups each consisting of five mice. 100 μg/mouse
of 341G2Ser (a solution in 200 μl of PBS containing 1% nude mouse
serum) was intravenously administered to the cancer-bearing mice once on
the 16th day, and the tumor size was measured until the 47th day. A human
anti-human serum albumin (HAS) antibody was used as a negative control.

(2) T24 Cells

[0196] A T24 cell mass that had undergone subcutaneous passage in the back
of nude mice three times were removed, and subcutaneously grafted into
the back of six-week-old female Balb/c nude mice (purchased from CLEA
Japan, Inc.). The tumor cell mass to be grafted is appropriately about 3
mm square. 10 days after the graft, the size of engrafted tumors was
measured. Cancer-bearing mice having a tumor size of 80 to 200 mm3
were classified into groups each consisting of five mice. 100 μg/mouse
of 341G2Ser (a solution in 200 μl of PBS containing 1% nude mouse
serum) was intravenously administered to the cancer-bearing mice once on
the 10th day, and the tumor size was measured until the 29th day. The
same amount of a human anti-DNP antibody was used as a negative control.

(3) Hs 766T Cells

[0197] 7×105 cells/mouse of Hs 766T cells were subcutaneously
grafted into the back of eight-week-old female Balb/c nude mice
(purchased from CLEA Japan, Inc.). 16 days after the graft, the size of
engrafted tumors was measured. Cancer-bearing mice having a tumor size of
50 to 140 mm3 were classified into groups each consisting of five
mice. 100 μg/mouse of 341G2Ser (a solution in 200 μl of PBS
containing 1% nude mouse serum) was intravenously administered to the
cancer-bearing mice once on the 16th day, and the tumor size was measured
until the 32nd day. The same amount of a human anti-DNP antibody was used
as a negative control.

(4) Capan-2 Cells

[0198] 2×106 cells/mouse of Capan-2 cells were subcutaneously
grafted into the back of six-week-old female Balb/c nude mice (purchased
from CLEA Japan, Inc.). 13 days after the graft, the size of engrafted
tumors was measured. Cancer-bearing mice having a tumor size of 30 to 130
mm3 were classified into groups each consisting of five mice. 10 or
100 μg/mouse of 341G2Ser (a solution in 200 μl of PBS containing 1%
nude mouse serum) was intravenously administered to the cancer-bearing
mice twice a week from the 13th day, and the tumor size was measured
until the 34th day. A human polyclonal antibody (hIgG) (manufactured by
Sigma Co.) was used as a negative control.

[0199] The tumor growth inhibition ratio (TGIR) was calculated from the
following formula.

100-[{(mean tumor volume of 341G2Ser-administered group on the last
measurement day-mean tumor volume of 341G2Ser-administered group on the
day of start of antibody administration)/(mean tumor volume of negative
control-administered group on the last measurement day-mean tumor volume
of negative control-administered group on the day of start of antibody
administration)}×100]

[0200] As a result, TGIR exceeded 100% in the T24, Hs766T and Capan-2
cancer-bearing mice, and a decrease in the tumor volume was observed in
the mice. On the other hand, TGIR was 73.4% in the Ramos cancer-bearing
mice, and an increase in the tumor volume was considerably suppressed in
the mice (Table 2). FIGS. 23 to 26 respectively show the change in the
tumor volume of cancer-bearing mice to which Ramos cells, T24 cells, Hs
766T cells and Capan-2 cells were respectively engrafted.

[0201] The inhibition ratio in each cell line is a value on the last
measurement day.

INDUSTRIAL APPLICABILITY

[0202] As shown in the Examples, the anti-CD40 antibody of the present
invention having a constant region into which a mutation is introduced
and an anti-CD40 antibody in which a part of the structure of the
subclass is substituted with that of another subclass have reduced ADCC
activity and CDC activity, while maintaining its activity. Accordingly,
the antibody of the present invention has the reduced cytotoxicity to
CD40-expressing cells when administered to a subject as a therapeutic
antibody, and thus can be used with safety.

[0203] All publications, patents and patent applications cited in this
specification are herein incorporated by reference in their entirety.